Whether apoptotic stimuli arise from the nucleus, cell membrane surface, or the mitochondria, ultimately, the stimuli converge on a process of activation of a family of cysteine proteases known as the caspases (cysteine aspartases). Activation of caspase family members mediate programmed cell death in normal physiology and a number of diseases, with caspase-3 standing at the center of the cell death program. Existing in a non-active pro-enzyme form in the cytosol of resting cells, caspase-3 is one key "effector" protease when activated. Thus, to monitor the final commitment of cells to death pathways, the need exists to directly quantify the enzymatic activity of caspase-3 in vivo. To meet this challenge, we designed and synthesized a class of peptide-based imaging agents that can penetrate the cell via non-receptor-mediated endocytic pathways, gaining access to the cytosolic compartment. These cell- penetrating optical imaging agents contain quenched fluorophores flanking target protease sequences which are cleaved and activated by caspase-3. Upon cleavage, these agents show caspase-3- dependent fluorescence signal amplification, thereby enabling high quality enzyme-specific molecular imaging of intracellular processes in vivo. Importantly, we discovered that upon intravitreal (intraocular) administration, our cell-penetrating peptides selectively accumulate in retinal ganglion cells (RGCs), the retinal neurons which are selectively injured and degenerate in glaucoma. RGCs are particularly accessible through an intravitreal approach, a routine ophthalmological procedure now performed everyday in the clinic. Thus, we plan to exploit this unanticipated and exciting cell-type-specific property of our peptides in retinal pathology for molecular imaging applications in ophthalmology. This renewal application is focused on translation of this strategy through advanced pre-clinical studies and development of a lead peptide. We propose pre-clinical testing in glaucoma models, toxicology testing, and metabolite profiling. We will advance a lead peptide into the clinic through pilot studies in a unique non-human primate model of glaucoma. These activities benefit from the combination expertise of this applicant team in chemistry, molecular imaging, biochemistry, and vision biology.

Public Health Relevance

Activation of enzymes that mediate programmed cell death occurs in normal physiology and in a number of diseases. One of these enzymes, known as caspase-3, stands at the center of the cell death program. Thus, to monitor the final commitment of cells to death pathways, the need exists to directly quantify the enzymatic activity of caspase-3 in vivo. To meet this challenge, we have designed and synthesized a new class of peptide- based imaging agents that can silently penetrate cells, gaining access to interior cellular compartments and be activated specifically by caspase-3 to emit visible fluorescence. We discovered that upon intraocular administration, our cell-penetrating peptides preferentially accumulate in retinal ganglion cells (RGCs), the retinal neurons which are selectively injured and degenerate in glaucoma Thus, we plan to translate this property of our peptides in retinal pathology for clinical molecular imaging applications. These activities benefit from the combination expertise of this applicant team in chemistry, molecular imaging, biochemistry, and vision biology.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY019587-14A1
Application #
8502936
Study Section
Clinical Molecular Imaging and Probe Development (CMIP)
Program Officer
Chin, Hemin R
Project Start
1999-07-01
Project End
2017-09-29
Budget Start
2013-09-30
Budget End
2014-09-29
Support Year
14
Fiscal Year
2013
Total Cost
$400,000
Indirect Cost
$150,000
Name
University of Texas MD Anderson Cancer Center
Department
None
Type
Other Domestic Higher Education
DUNS #
800772139
City
Houston
State
TX
Country
United States
Zip Code
77030