Cystine-knot peptides: emerging tools for cancer imaging and therapy

Abstract

Cystine-knot miniproteins, also known as knottins, constitute a large family of structurally related peptides with diverse amino acid sequences and biological functions. Knottins have emerged as attractive candidates for drug development as they potentially fill a niche between small molecules and protein biologics, offering drug-like properties and the ability to bind to clinical targets with high affinity and selectivity. Due to their extremely high stability and unique structural features, knottins also demonstrate promise in addressing challenging drug development goals, including the potential for oral delivery and the ability to access intracellular drug targets. Several naturally-occurring knottins have recently received approval for treating chronic pain and irritable bowel syndrome, while others are under development for tumor imaging applications. To expand beyond nature’s repertoire, rational and combinatorial protein engineering methods are generating tumor-targeting knottins for use as cancer diagnostics and therapeutics.

Keywords::

• disulfide-rich peptides
• drug discovery
• knottin
• miniprotein
• molecular imaging
• protein engineering
• therapeutics
• tumor targeting

Acknowledgement

The authors acknowledge fellowship support from the following sources: Stanford Department of Bioengineering and Stanford Bio-X Fellowship Programs (SE Ackerman), the Stanford Child Health Research Institute and the Anne T. and Robert M. Bass Endowed Fellowship in Pediatric Cancer and Blood Diseases (NV Currier), and Lucile Packard Foundation for Children’s Health and the Stanford NIH/NCRR CTSA award number UL1 RR025744 (JM Bergen).

Financial & competing interests disclosure

Jennifer Cochran is an inventor on Stanford-owned intellectual property and has funded grants related to her group's work on knottin peptides. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• Knottins are small (30–50 amino acid), constrained peptides that show promise as diagnostics and therapeutics due to their innate thermal and proteolytic stability.

• The cystine-knot motif is the main driver of stability in this class of peptides; however, cyclization of knottin peptides by connecting their N- and C-termini can sometimes confer increased stability and oral bioavailability.

• Nature serves as a rich source of new knottin-based drugs, but rational and combinatorial methods are also being used to develop knottins that bind to tumor-associated biomarkers and other clinical targets.

• Natural knottin peptides have been approved for treating indications such as chronic pain and irritable bowel syndrome, with other knottin drug candidates in the clinical pipeline.

• High affinity tumor-targeting knottins have ideal pharmacokinetic properties for molecular imaging applications, namely rapid tumor uptake and blood clearance, which results in high tumor-to-normal tissue imaging contrast.

• Some natural knottins are internalized into cells; this attribute allowed an engineered cyclotide to access and modulate an intracellular cancer target.

• Future challenges include engineering knottins against a broader range of cancer-related targets and tuning their pharmacokinetic and pharmacodynamic properties for therapeutic applications.