Noncanonical Amino Acids to Improve the pH Response of pHLIP Insertion at Tumor Acidity

Abstract

The pH-Low Insertion Peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membrane with a pH 50 of 6.1 while most solid tumors have extracellular pH (pH e ) of 6.5-7.0. To close this gap, a ** We thank Prof. Dr. Donald M. Engelman (Yale) for discussion and support; Prof. Dr. Eriks Rozners and Prof. Dr. Susan Bane (both of SUNY-Binghamton) for frequent use of their fluorimeter and plate reader, respectively. We also thank Raemer J. Lapid (for assistance in processing data), Rebecca A. Chandler, Meghan M. Bell, Vladyslav Nazarenko, Ilana G. Bandler (for assistance in cell assays) and Emma A. Gordon (for assistance in synthesis). This work was supported by SUNY-Binghamton University (BU) (Start-up funds to M.A. and L.Y., various financial supports to J.O., L.K. and M.C., HHMI-BU undergraduate summer fellowships to C-H. E. and R. L.), NIH (R01-GM073857 for training of M.A. and initial work), and NSF grant CHE-0922815 for the Regional NMR Facility (600 MHz instrument) at SUNY-Binghamton.

Therapeutic Efficacy of a Family of pHLIP–MMAF Conjugates in Cancer Cells and Mouse Models

“…15–18 For example, the apparent pH 50 of insertion across 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membrane for pHLIP(WT) is about 6.1 (Table 1). 11,19–21 Of the six pHLIP variants considered here, three have a larger pH 50 than pHLIP(WT): (1) pHLIP(D25E), where Asp25 is substituted by the more hydrophobic glutamic acid, 20 (2) pHLIP(P20G), where the Pro to Gly substitution increases pH 50 by enhancing the propensity of the peptide to form an α -helix, 22 and (3) pHLIP(D14Gla;D25Aad), where Asp14 is substituted by an γ -carboxyglutamic acid (Gla), which has an additional side chain carboxylic acid, and where Asp25 is substituted for the more hydrophobic α -aminoadipic acid (Aad). This variant not only shows an increase in pH 50 (6.79) but also a slight increase in insertion cooperativity (i.e., sharper pH-response).…”

“…This variant not only shows an increase in pH 50 (6.79) but also a slight increase in insertion cooperativity (i.e., sharper pH-response). 11 While the variants with increased pH 50 of insertion might match better the tumor microenvironment pH, one cannot exclude that a decreased pH 50 of insertion may provide better selectivity in vivo by increasing pHLIP’s sensitivity to acidic environments, thereby decreasing off-targeted delivery. It is therefore not clear, at this very early stage of the development of pHLIP as a therapeutic, whether a high or low pH 50 is preferable for effective targeting and delivery in vivo.…”

Affinity-controlled protein encapsulation into sub-30 nm telodendrimer nanocarriers by multivalent and synergistic interactions

“…We would like to emphasize that the protein release rate by serum protein exchange can vary for different cargo proteins and can be tailored by engineering the relative binding affinity of telodendrimer to the cargo protein. [19] For example, insulin, a pH-low insertion peptide (pHLIP-Alxa750-DOX)[45] conjugated with doxorubicin and a near infrared dye Alexa750, and granulocyte-colony stimulating factor (GCSF) loaded in the telodendrimer NPs were very stable against BSA incubation at a biological concentration of 40 mg/mL (Fig. S26–S29), indicating strong binding affinity between these cargo proteins and telodendrimers.…”

“…Bovine insulin ( M w 5.8 kDa, isoelectric point 5.7) was purchased from Gemini Bio-Products. WT pH low insertion peptide conjugated with near infrared dye (Alexa 750) and drug (doxorubicin), which is named as pHLIP-Alxa750-DOX ( M w ~6 kDa, isoelectric point ~4), was provided by Prof. Ming An of Department of Chemistry at SUNY Binghamton University, details can be found in the previous publication[45]. Granulocyte-colony stimulating factor (GCSF, M w 18.8 kDa, isoelectric point 5.7) was provided by Prof. Li-Ru Zhao of Department of Neurosurgery at SUNY Upstate Medical University, which was originally purchased from Amgen.…”

Stimuli-responsive nanoparticles for targeting the tumor microenvironment

“…However improvements on the transition pH have been made as a result of an extensive structure–activity relationship study for a series of peptide variants performed by the An group [69]. They found that by replacing specific amino acid residues within the transmembrane domain, the transition pH can be tuned from 6.1 to 6.9.…”