Polymer-Tethered Quenched Fluorescent Probes for Enhanced Imaging of Tumor-Associated Proteases.

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become the standard of care in cancer surgeries. One of the key parameters to optimize in contrast agents is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker, as well as the positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased overall signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

Polymer-tethered quenched fluorescent probes for enhanced imaging of tumor associated proteases.

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

Polymer-based antibody mimetics (iBodies) target human PD-L1 and function as a potent immune checkpoint blocker.

Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability. Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.

Identification of specific carbonic anhydrase inhibitors via in situ click chemistry, phage-display and synthetic peptide libraries: comparison of the methods and structural study.

The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.

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

Human cathepsin D (CatD), a pepsin-family aspartic protease, plays an important role in tumor progression and metastasis. Here, we report the development of biomimetic inhibitors of CatD as novel tools for regulation of this therapeutic target. We designed a macrocyclic scaffold to mimic the spatial conformation of the minimal pseudo-dipeptide binding motif of pepstatin A, a microbial oligopeptide inhibitor, in the CatD active site. A library of more than 30 macrocyclic peptidomimetic inhibitors was employed for scaffold optimization, mapping of subsite interactions, and profiling of inhibitor selectivity. Furthermore, we solved high-resolution crystal structures of three macrocyclic inhibitors with low nanomolar or subnanomolar potency in complex with CatD and determined their binding mode using quantum chemical calculations. The study provides a new structural template and functional profile that can be exploited for design of potential chemotherapeutics that specifically inhibit CatD and related aspartic proteases.

The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic hydrolase acting mostly on nonhistone protein substrates. Investigations of the substrate specificity of HDAC6 are confounded by the presence of 2 catalytically active deacetylase domains (DD1 and DD2). In this study, acetylome peptide microarrays and peptide libraries were used to map the substrate specificity of DD1 and DD2 of human HDAC6. The results show that DD1 is solely responsible for the deacetylation of substrates harboring the acetyllysine at their C terminus, whereas DD2 exclusively deacetylates peptides with an internal acetyllysine residue. Also, statistical analysis of the deacetylation data revealed amino acid preferences at individual positions flanking the acetyllysine, where glycine and arginine residues are favored at positions N-terminal to the central acetyllysine; negatively charged glutamate is strongly disfavored throughout the sequence. Finally, the deacylation activity of HDAC6 was profiled by using a panel of acyl derivatives of the optimized peptide substrate and showed that HDAC6 acts as a proficient deformylase. Our data thus offer a detailed insight into the substrate preferences of the individual HDAC6 domains at the peptide level, and these findings can in turn help in elucidating the biologic roles of the enzyme and facilitate the development of new domain-specific inhibitors as research tools or therapeutic agents.-Kutil, Z., Skultetyova, L., Rauh, D., Meleshin, M., Snajdr, I., Novakova, Z., Mikesova, J., Pavlicek, J., Hadzima, M., Baranova, P., Havlinova, B., Majer, P., Schutkowski, M., Barinka, C. The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.