It is a central idea of the NIH that basic research will lead to new approaches in medicine, and we believe that we have found one. As a result of earlier funding of this grant, we have discovered a peptide that (1) targets acidic tissues in vivo, including tumors, (2) can deliver polar molecules into cells, releasing them in the cytoplasm, and (3) gives an opportunity to better understand how peptides can insert across membranes. We now plan to explore both the basic and applied aspects of this discovery. The peptide, which we call """"""""pHLIP"""""""" (for pH (Low) Insertion Peptide) is soluble as an unstructured monomer in aqueous solution, binds as an unstructured monomer to the surface of a bilayer or membrane, and inserts across the bilayer as a trans-membrane helix (TM) when the pH is lowered. We have established the basic energetics and kinetics of peptide insertion. We have shown that a labeled version of pHLIP targets and images tumors as small as 1 mm in mice, and that the imaging accurately identifies tumor borders. We have also established that large, polar cargo molecules (M ~ 1000 Da, log P ~ -2) attached to the inserting end of pHLIP by a disulfide are delivered across membranes and released in the cytoplasms of cultured tumor cells at low pH. By continuing our basic research we hope to frame the technology for use in the clinic. We will study the process of and sequence requirements for insertion of water soluble peptides into membranes, find improved ways to target tumors and other acidic tissues, and develop expanded ways to deliver polar molecules into cells, releasing them into the cytoplasm by disulfide or ester cleavage. Targeting imaging agents to tumors with pHLIP could aid in diagnosis or act as a guide for surgery, and delivering therapeutics could assist in treatment. Using biophysical, biochemical, and biological approaches, we will seek answers to the following questions: 1. What are the kinetic intermediates, energetics and structures of the bilayer and peptide during insertion? 2. Which sequence features allow a water-soluble peptide to insert spontaneously to form a TM? 3. What role(s) do lipids play in TM insertion? 4. Can pHLIP be used to image cargo delivery in vivo? 5. What is the range of polar molecules that can be delivered to cells?

Public Health Relevance

We have discovered a new way to target tumors based on their acidity, using a small peptide that inserts across the membranes of the cells in the tissue. The peptide accumulates preferentially in the tumor, which is acidic as a result of its metabolism, and we can put a label on the peptide to image the tumor for diagnosis or to assist a surgeon in removing it by showing where its boundaries are. The peptide can also be used to deliver drugs into the cells of the tumor, so improved therapy might be possible.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073857-07
Application #
8488443
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2006-05-15
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
7
Fiscal Year
2013
Total Cost
$469,941
Indirect Cost
$102,962
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Golijanin, Jovana; Amin, Ali; Moshnikova, Anna et al. (2016) Targeted imaging of urothelium carcinoma in human bladders by an ICG pHLIP peptide ex vivo. Proc Natl Acad Sci U S A 113:11829-11834
Narayanan, Theyencheri; Weerakkody, Dhammika; Karabadzhak, Alexander G et al. (2016) pHLIP Peptide Interaction with a Membrane Monitored by SAXS. J Phys Chem B 120:11484-11491
Svoronos, Alexander A; Engelman, Donald M; Slack, Frank J (2016) OncomiR or Tumor Suppressor? The Duplicity of MicroRNAs in Cancer. Cancer Res 76:3666-70
Weerakkody, Dhammika; Moshnikova, Anna; El-Sayed, Naglaa Salem et al. (2016) Novel pH-Sensitive Cyclic Peptides. Sci Rep 6:31322
Anderson, Michael; Moshnikova, Anna; Engelman, Donald M et al. (2016) Probe for the measurement of cell surface pH in vivo and ex vivo. Proc Natl Acad Sci U S A 113:8177-81
Tapmeier, Thomas T; Moshnikova, Anna; Beech, John et al. (2015) The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions. Proc Natl Acad Sci U S A 112:9710-5
Reshetnyak, Yana K (2015) Imaging Tumor Acidity: pH-Low Insertion Peptide Probe for Optoacoustic Tomography. Clin Cancer Res 21:4502-4
Onyango, Joab O; Chung, Michael S; Eng, Chee-Huat et al. (2015) Noncanonical amino acids to improve the pH response of pHLIP insertion at tumor acidity. Angew Chem Int Ed Engl 54:3658-63
Heim, Erin N; Marston, Jez L; Federman, Ross S et al. (2015) Biologically active LIL proteins built with minimal chemical diversity. Proc Natl Acad Sci U S A 112:E4717-25
Deacon, John C; Engelman, Donald M; Barrera, Francisco N (2015) Targeting acidity in diseased tissues: mechanism and applications of the membrane-inserting peptide, pHLIP. Arch Biochem Biophys 565:40-8

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