The success of protein-based therapeutics is revolutionizing drug development. Unlike small molecule drugs, peptide and protein-based therapeutics can target with high selectivity and specificity defective protein-protein interaction involved in human disease. Despite their success, however, there are still numerous stability and delivery issues associated with their use as therapeutic agents. For example, monoclonal antibodies (one the most successful protein-based therapeutics with several blockbuster drugs on the market and many more in clinical development) can only target extracellular molecular targets due to their inability to cross biological membranes. They are also extremely expensive to produce and are not bioavailable due to their susceptibility to proteolytic degradation. These issues have led to the exploration of alternative protein scaffolds as a source for novel types of protein-based therapeutics. In response to this important challenge, we propose the use of genetically- and chemically-encoded libraries of cyclotides for selecting specific cyclotide sequences able to modulate protein-protein interactions. Cyclotides are a new emerging family of large plant-derived backbone-cyclized polypeptides (?30 amino acids long) that share a 3 disulfide-stabilized core characterized by an unusual knotted structure. They have several characteristics that make them ideal drug development tools. To achieve this objective we propose to use protein-splicing technology developed in the Camarero lab to generate large, genetically encoded cyclotide libraries inside live bacterial cells. These cell-based libraries wil be screened using different in-cell reporters to identify bacteria encoding active cyclotide sequences. In addition, we will also develop novel strategies for the generation and rapid screening of chemically synthesized cyclotides targeted against membrane associated extracellular receptors. Selected cyclotides will be characterized and assayed in mammalian cells to test their ability to antagonize the selected intracellular or extracellular protein targes. We will also explore the cell penetrating properties of these interesting microproteins as well as ways to improve it. Finally, we also want to study their pharmacokinetic (PK) properties and explore different approaches to improve their bioavailability.

Public Health Relevance

This proposal describes innovative approaches for the screening and selection of a novel class of protein-based therapeutics (cyclotides). We will employ both genetically- and chemically-encoded cyclotide-based libraries to screen intracellular and extracellular protein-protein interactions using high throughput screening methods. It is anticipated that these combined approaches will have profound implications for human health and the way protein-based therapeutics can be used in the future to target specific extra- and intracellular protein-protein interactions involved in the development of cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM113636-01A1
Application #
8987156
Study Section
Special Emphasis Panel (ZRG1-BCMB-W (02))
Program Officer
Fabian, Miles
Project Start
2015-08-01
Project End
2019-04-30
Budget Start
2015-08-01
Budget End
2016-04-30
Support Year
1
Fiscal Year
2015
Total Cost
$325,875
Indirect Cost
$128,375
Name
University of Southern California
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Bi, Tao; Li, Yilong; Shekhtman, Alexander et al. (2018) In-cell production of a genetically-encoded library based on the ?-defensin RTD-1 using a bacterial expression system. Bioorg Med Chem 26:1212-1219
Jagadish, Krishnappa; Camarero, Julio A (2017) Recombinant Expression of Cyclotides Using Split Inteins. Methods Mol Biol 1495:41-55
Lesniak, Wojciech G; Aboye, Teshome; Chatterjee, Samit et al. (2017) In vivo Evaluation of an Engineered Cyclotide as Specific CXCR4 Imaging Reagent. Chemistry 23:14469-14475
Gould, Andrew; Camarero, Julio A (2017) Cyclotides: Overview and Biotechnological Applications. Chembiochem 18:1350-1363
Camarero, Julio A (2017) Cyclotides, a versatile ultrastable micro-protein scaffold for biotechnological applications. Bioorg Med Chem Lett 27:5089-5099
Li, Yilong; Gould, Andrew; Aboye, Teshome et al. (2017) Full Sequence Amino Acid Scanning of ?-Defensin RTD-1 Yields a Potent Anthrax Lethal Factor Protease Inhibitor. J Med Chem 60:1916-1927
Borra, Radhika; Camarero, Julio A (2017) Protein Chemical Modification Inside Living Cells Using Split Inteins. Methods Mol Biol 1495:111-130
Li, Yilong; Aboye, Teshome; Breindel, Leonard et al. (2016) Efficient recombinant expression of SFTI-1 in bacterial cells using intein-mediated protein trans-splicing. Biopolymers 106:818-824
Aboye, Teshome; Meeks, Christopher J; Majumder, Subhabrata et al. (2016) Design of a MCoTI-Based Cyclotide with Angiotensin (1-7)-Like Activity. Molecules 21:152
Aboye, Teshome; Kuang, Yuting; Neamati, Nouri et al. (2015) Rapid parallel synthesis of bioactive folded cyclotides by using a tea-bag approach. Chembiochem 16:827-33

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