A Knotty Challenge: Novel cystine-knot peptide inhibitors of HTRA1
The July BPS journal club features an article showcasing the power of cystine-knot peptides (CKPs) as high-affinity and high-specificity protein inhibitors. A team from the laboratories of Rami Hannoush and Daniel Kirchhofer at Genentech created diversity libraries based on the carboxypeptidase A1 inhibitor and Ecballium elaterium trypsin inhibitor II CKPs. They performed phage display and affinity maturation studies to select potent inhibitors of a different serine protease, HTRA1 (https://doi.org/10.1038/s41467-024-48655-w)1. The authors then characterized their modes of binding in structural studies and exploited differences in this binding pocket between serine protease family members to increase selectivity to the target. Specificity is an especially challenging problem in the serine protease family due to the large number of related family members with a fairly conserved binding pocket, including four highly related members of the HTRA family, HTRA1-4.
HTRA1, or high-temperature requirement A serine peptidase 1, is a trimeric serine protease implicated in several disorders, including arthritis, osteoporosis, age-related macular degeneration, Alzheimer’s, and chemoresistance. Both antibody-based (https://doi.org/10.1073/pnas.1917608117)2 and small molecule, peptidomimetic-like inhibitors of HTRA1 (https://doi.org/10.1016/j.bmcl.2024.129814)3 have been identified, and clinical studies have been initiated with a Fab inhibitor of HTRA1 for age-related macular degeneration (NCT03972709).
Cystine-knots are structural motifs in proteins comprising three disulfide bridges between cysteine amino acids, where one strand or loop passes through another loop, forming a rotaxane structure that is protease resistant and shows both chemical and thermal stability. The core portions of CKPs have relatively low molecular weights (generally around 30 amino acids and less than 4 kDa) and are naturally occurring in animals, plants, and insects. Some common examples include nerve growth factor, transforming growth factor-beta, and various venom peptides from snails, spiders, and scorpions. They can also be chemically synthesized or manufactured using biological expression systems, making them excellent candidates for therapeutics.
The authors generated phage peptide libraries by introducing amino acid randomization and length variation into the surface-exposed loops of the CKP scaffolds and screened the resulting phage for binding to HTRA1 constructs by phage ELISA. Hits from the initial screen were modified through affinity maturation studies, and several inhibitors were identified with single-digit nanomolar affinities that also showed IC50s for HTRA1 activity on a substrate in the same range. Biochemical studies were then used to map the approximate binding regions for the compounds on HTRA1, followed by structural studies that demonstrated that the CKP inhibitors bind a cryptic pocket near the active site and stabilize a structure that renders HTRA1 non-competent for protease activity. This cryptic pocket showed some differences from other members of the HTRA protease family, and the authors used a combination of structure-based design and further affinity maturation to exploit these differences and generate inhibitors with both high potency and high selectivity for HTRA1, even when compared to HTRA2, 3, and 4.
This article is a fantastic example of collaborative, multidisciplinary work using genetics, biochemistry, and structural biology to identify peptide inhibitors with high affinity and selectivity for a challenging target. Furthermore, it is another example of optimizing naturally occurring structures to develop candidate therapeutics that have the potential to be stable and well-tolerated since there are numerous examples of CKPs in biology. I look forward to seeing the effects of these inhibitors in animal models of diseases related to HTRA1 in the near future, as well as publications using a similar approach to identify cystine-knot peptide inhibitors of other challenging targets.
Dave Garman, PhD
Member, BPF Scientific Advisory Board
1Li, Yanjie et al. Cystine-Knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region. Nature Communications 15:4359 (2024)
2Tom, Irene et al. Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy. PNAS 117(18):9952-63 (2020)
3Dennis, David G et al. Identification of highly potent and selective HTRA1 inhibitors. Bioorganic & Medicinal Chemistry Letters 109, September 2024 (available online ahead of print)
Read previous editions of the BPF Journal Club series: https://www.boulderpeptide.org/journal-club