Single‐ and Double‐Headed Chemical Probes for Detection of Active Cathepsin D in a Cancer Cell Proteome

Abstract

Activity-based proteomics employing active-site-directed chemical probes for enzymatic activity profiling in complex proteomes has greatly accelerated the functional annotation of proteins. [1,2] In protease research, new generations of activitybased probes have led to tremendous progress in our understanding of the biochemistry and physiology of cysteine peptidases over the last decade. [3,4] These probes were designed for in vitro applications as well as for in vivo monitoring in living cells and whole animals, and has directed research towards biomarker discovery and drug screening. However, the current availability of analogous chemical tools selective for other classes of peptidases is limited. The situation is especially critical in the case of aspartic peptidases, despite the fact that these enzymes play an important role in a number of devastating human pathologies, such as AIDS, Alzheimer's disease, and cancer. A major reason for the limited number of available probes for aspartic peptidases is the lack of suitable covalent inhibitors that can be used as starting templates for probe derivation. There are only a few examples of recent advances in this field, including probes for human g-secretase and malaria plasmepsins that were constructed based on the concept of photoaffinity labeling. [5,6] In this work, we focus on cathepsin D, an aspartic peptidase that is critically associated with cancer development and progression through its complex proteolytic and proliferative action and is a prognostic marker in breast cancer (for a recent review, see ref. [7]). There is still insufficient knowledge regarding the interaction partners and activity regulation mechanisms of this therapeutic target, and the introduction of novel imaging tools is required for these studies.Here, we present a convenient synthesis of novel proteomicactivity-based probes for cathepsin D that are suitable for applications in activity profiling and analysis of post-translational activation processing.The design of three probes, sBAP-09, dBAP-09, and FAP-09, is shown in Figure 1. Their structures consist of three functionalities: 1) a peptidic binding core, 2) a photoreactive group as a probe "warhead", and 3) a fluorescent group or biotin as a reporter tag. The binding core was constructed by using pepstatin, a reversible peptidomimetic inhibitor of aspartic peptidases, as a template. The pepstatin molecule was structurally minimized and optimized for cathepsin D inhibition [8] to yield the binding core sequence shown in Figure 1. The statin residue in the peptidic core interacts with the enzyme's catalytic residues and acts as a transition-state mimic. The binding core scaffold was modified at its N and/or C terminus to incorporate functionalities 2) and 3). The crystal structure of human cathepsin D in complex with pepstatin (PDB ID: 1LYB) suggests that bulky substituents in the terminal positions will protrude from the enzyme active site and should not interfere with the interaction of ligand and enzyme. In the sBAP-09 and dBAP-09 p...

Pepstatin-Based Probes for Photoaffinity Labeling of Aspartic Proteases and Application to Target Identification

“…In this work, we developed clickable probes based on pepstatin to profile aspartic proteases by PAL. Although some studies have reported solid phase synthesis of aspartic protease probes, these made use of custom-synthesized building blocks or placed bulky cross-linkers at a distal position from the inhibitory scaffold that binds the active site cleft . We here showed that a minimal diazirine linker is suitable for incorporation into the peptide sequence of the general aspartic protease inhibitor pepstatin.…”

“…Although some studies have reported solid phase synthesis of aspartic protease probes, these made use of custom-synthesized building blocks 19 or placed bulky cross-linkers at a distal position from the inhibitory scaffold that binds the active site cleft. 18 We here showed that a minimal diazirine linker is suitable for incorporation into the peptide sequence of the general aspartic protease inhibitor pepstatin. Conveniently, the probes can be synthesized entirely on solid support by using commercially available building blocks.…”

“…These probes are sometimes referred to as affinity-based probes (AfBPs) and have been regularly applied in photoaffinity labeling (PAL) experiments. Multiple examples of such AfBPs exist for metalloproteases, most of which are based on Zn 2+ -coordinating peptide hydroxamate inhibitors. − Although various AfBPs have been successfully designed for the intramembrane proteases γ-secretase and signal peptide peptidase (SPP), , the toolbox for soluble aspartic proteases is very limited. − …”

Biomimetic Macrocyclic Inhibitors of Human Cathepsin D: Structure–Activity Relationship and Binding Mode Analysis

“…1 H NMR (400 MHz, DMSO-d 6 ): 0.84 (3H, d, J = 6.8), 0.85 (3H, d, J = 6.8), 1.06−1.73 (31H, m), 1.97−2.05 (1H, m), 2.15 (1H, dd, J = 14.2, 6.3), 2.19 (2H, dd, J = 6.3, 6.1), 2.28 (1H, dd, J = 14.2, 7.8), 2.71−2.79 (1H, m), 3.28−3.37 (1H, m), 3.74−3.81 (1H, m), 3.93 (1H, dd, J = 8.0, 5.9), 3.82−3.87 (1H, m), 4.80 (1H, d, J = 5.5), 7.45 (1H, d, J = 9.2), 8.02 (1H, dd, J = 7.1, 4.4). 13 (3S,7S,8S)-7-Hydroxy-8-(4-hydroxybenzyl)-3-isopropyl-1,4,9-triazacyclohenicosane-2,5,10-trione (20). 1 H NMR (400 MHz, DMSOd 6 ): 0.84 (6H, d, J = 6.9), 1.02−1.46 (18H, m), 1.94−2.03 (1H, m), 2.03−2.10 (1H, m), 2.10−2.17 (1H, m), 2.21 (1H, dd, J = 14.1, 6.8), 2.26 (1H, dd, J = 14.1, 6.9), 2.57 (1H, dd, J = 13.9, 10.2), 2.69−2.77 (2H, m), 3.26−3.34 (1H, m), 3.81 (1H, dddd, J = 9.7, 9.5, 4.7, 1.6), 3.92−3.98 (3H, m), 6.59−6.63 (2H, m), 6.95−7.00 (2H, m), 7.58 (1H, br s), 7.60 (1H, d, J = 1.8), 7.96 (1H, dd, J = 7.0, 4.4), 9.05 (1H, br s).…”

“…This linear pseudo-pentapeptide originally isolated from actinobacteria is effective against many aspartic proteases from the pepsin family with inhibition constants as low as 10 –10 M. Its potency is attributable to the nonproteinogenic amino acid statine [(3 S ,4 S )-4-amino-3-hydroxy-6-methylheptanoic acid], the hydroxyl group of which interacts with catalytic aspartate residues and acts as a transition-state analogue. Pepstatin A is widely used as a research tool for enzyme affinity purification, active site titration, imaging, and structural mapping of binding sites. − However, its use as a therapeutic is limited due to its large size and unfavorable physicochemical properties including poor solubility, which are reflected in its low bioavailability and rapid clearance …”