Molecularly targeted protease-activated probes for visualization of glioblastoma: a comparison with 5-ALA.

OBJECTIVE: The highly infiltrative growth of glioblastoma (GBM) makes distinction between the tumor and normal brain tissue challenging. Therefore, fluorescence-guided surgery is often used to improve visual identification of radiological tumor margins. The aim of this study was to evaluate the ability of recently developed molecularly targeted near-infrared (NIR) protease-activated probes to visualize GBM tissue and to compare the most promising candidate with the gold standard, 5-aminolevulinic acid (5-ALA). METHODS: Single-substrate probes 6QC-ICG and 6QC-Cy5 (cysteine cathepsin cleavable), double-substrate probes AG2-FNIR and AG2-Cy5 (cysteine cathepsin and caspase 3 cleavable), and 5-ALA were administered intravenously to mice with orthotopic tumors. Activation of the probes was also evaluated in cell cultures in vitro and in biopsy material from patients with GBM ex vivo. The tumor to normal brain tissue fluorescence ratio (TNR) was quantified in brain sections using preclinical and clinical visualization platforms, and in tissue homogenates and cell suspensions using spectrofluorimetry. Subcellular localization of the fluorophores was visualized by confocal microscopy. RESULTS: In vitro, the single-substrate probe 6QC-ICG was cleaved in glioma cells and macrophages, and the resulting fluorophore accumulated intracellularly. In experimental GBMs, both single- and double-substrate probes visualized tumor tissue, while in healthy brain tissue the signal was minimal. TNR was highest for 6QC-ICG and AG2-FNIR, but the signal intensity was higher for 6QC-ICG. Using xenograft and syngeneic mouse models, as well as human GBM biopsy material ex vivo, the authors confirmed the ability of 6QC-ICG to specifically visualize the glioma tissue using preclinical and clinical visualization platforms. Finally, a comparison with 5-ALA in animals coadministered with both compounds revealed a higher TNR for 6QC-ICG in experimental GBMs. CONCLUSIONS: The cysteine cathepsin-cleavable probe 6QC-ICG is activated by glioma cells and tumor-associated macrophages, leading to a high contrast between tumor and nontumorous brain tissue that is superior to that of the current standard, 5-ALA. In addition to a well-defined mechanism of action, protease-activated probes that use NIR fluorophores (e.g., indocyanine green) have the advantage of low absorption and scattering of the NIR light and lower tissue autofluorescence. These results suggest that 6QC-ICG has the potential to become the targeted agent in intraoperative detection of GBM tissue using fluorescence imaging.

Recent advances in ratiometric fluorescence imaging of enzyme activity in vivo.

Among molecular imaging modalities that can monitor enzyme activity in vivo, optical imaging provides sensitive, molecular-level information at low-cost using safe and non-ionizing wavelengths of light. Yet, obtaining quantifiable optical signals in vivo poses significant challenges. Benchmarking using ratiometric signals can overcome dependence on dosing, illumination variability, and pharmacokinetics to provide quantitative in vivo optical data. This review highlights recent advances using fluorescent probes that are processed by enzymes to induce photophysical changes that can be monitored by ratiometric imaging. These diverse strategies include caged fluorophores that change photophysical properties upon enzymatic cleavage, as well as multi-fluorophore systems that are triggered by enzymatic cleavage to alter optical outputs in one or more fluorescent channels. The strategies discussed here have great potential for further development as well as potential broad applications for targeting diverse enzymes important for a wide range of human diseases.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections.

Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here, we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologues in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling, and mouse models of infection, we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes and validate FphE as a target for the development of imaging contrast agents for the rapid detection of S. aureus infections.

Mixed Alkyl/Aryl Phosphonates Identify Metabolic Serine Hydrolases as Antimalarial Targets.

Malaria, caused by Plasmodium falciparum, remains a significant health burden. A barrier for developing anti-malarial drugs is the ability of the parasite to rapidly generate resistance. We demonstrated that Salinipostin A (SalA), a natural product, kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism with a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent anti-parasitic potencies which enabled identification of therapeutically relevant targets. We also confirm that this compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor, Orlistat. Like SalA, our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are a promising, synthetically tractable anti-malarials with a low-propensity to induce resistance.

Covalent Macrocyclic Proteasome Inhibitors Mitigate Resistance in Plasmodium falciparum.

The Plasmodium proteasome is a promising antimalarial drug target due to its essential role in all parasite lifecycle stages. Furthermore, proteasome inhibitors have synergistic effects when combined with current first-line artemisinin and related analogues. Linear peptides that covalently inhibit the proteasome are effective at killing parasites and have a low propensity for inducing resistance. However, these scaffolds generally suffer from poor pharmacokinetics and bioavailability. Here we describe the development of covalent, irreversible, macrocyclic inhibitors of the Plasmodium falciparum proteasome. We identified compounds with excellent potency and low cytotoxicity; however, the first generation suffered from poor microsomal stability. Further optimization of an existing macrocyclic scaffold resulted in an irreversible covalent inhibitor carrying a vinyl sulfone electrophile that retained high potency and low cytotoxicity and had acceptable metabolic stability. Importantly, unlike the parent reversible inhibitor that selected for multiple mutations in the proteasome, with one resulting in a 5,000-fold loss of potency, the irreversible analogue only showed a 5-fold loss in potency for any single point mutation. Furthermore, an epoxyketone analogue of the same scaffold retained potency against a panel of known proteasome mutants. These results confirm that macrocycles are optimal scaffolds to target the malarial proteasome and that the use of a covalent electrophile can greatly reduce the ability of the parasite to generate drug resistance mutations.

Chemoproteomic identification of a DPP4 homolog in Bacteroides thetaiotaomicron.

Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.

Protease Activated Probes for Real-Time Ratiometric Imaging of Solid Tumors.

Surgery is the preferred treatment option for most solid tumors. However, inaccurate detection of cancer borders leads to either incomplete removal of malignant cells or excess excision of healthy tissue. While fluorescent contrast agents and imaging systems improve tumor visualization, they can suffer from low signal-to-background and are prone to technical artifacts. Ratiometric imaging has the potential to eliminate many of these issues such as uneven probe distribution, tissue autofluorescence, and changes in positioning of the light source. Here, we describe a strategy to convert quenched fluorescent probes into ratiometric contrast agents. Conversion of the cathepsin-activated probe, 6QC-Cy5, into a two-fluorophore probe, 6QC-RATIO, significantly improved signal-to-background in vitro and in a mouse subcutaneous breast tumor model. Tumor detection sensitivity was further enhanced using a dual-substrate AND-gate ratiometric probe, Death-Cat-RATIO, that fluoresces only after orthogonal processing by multiple tumor-specific proteases. We also designed and built a modular camera system that was coupled to the FDA-approved da Vinci Xi robot, to enable real-time imaging of ratiometric signals at video frame rates compatible with surgical workflows. Our results demonstrate that ratiometric camera systems and imaging probes have the potential to be clinically implemented to improve surgical resection of many types of cancer.

Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome.

The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic ß2 and ß5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the ß5 P1 binding pocket, compromising compound binding. Dual targeting of P. falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria.

Solid Phase Synthesis of Fluorosulfate Containing Macrocycles for Chemoproteomic Workflows.

Macrocyclic peptides are attractive for chemoproteomic applications due to their modular synthesis and potential for high target selectivity. We describe a solid phase synthesis method for the efficient generation of libraries of small macrocycles that contain an electrophile and alkyne handle. The modular synthesis produces libraries that can be directly screened using simple SDS-PAGE readouts and then optimal lead molecules applied to proteomic analysis. We generated a library of 480 macrocyclic peptides containing the weakly reactive fluorosulfate (OSF) electrophile. Initial screening of a subset of the library containing each of the various diversity elements identified initial molecules of interest. The corresponding positional and confirmational isomers were then screened to select molecules that showed specific protein labeling patterns that were dependent on the probe structure. The most promising hits were applied to standard chemoproteomic workflows to identify protein targets. Our results demonstrate the feasibility of rapid, on-resin synthesis of diverse macrocyclic electrophiles to generate new classes of covalent ligands.

Finding optimal drug target sites in parasite pathogens.

Benns et al. have recently combined a chemoproteomic profiling method with a CRISPR-based gene-editing method to identify chemically targetable residues essential for fitness in the parasite Toxoplasma gondii. The result is a strategy that enables rapid discovery of new drug targets to combat T. gondii and other related parasites.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of S. aureus Infections.

Staphylococcus aureus is a major human pathogen responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here we describe the development of oxadiazolonebased activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologs in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling and mouse models of infection we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes (ABPs) and validate FphE as a target for development of imaging contrast agents for the rapid detection of S. aureus infections.

Trypanosoma brucei Acyl-Protein Thioesterase-like (TbAPT-L) Is a Lipase with Esterase Activity for Short and Medium-Chain Fatty Acids but Has No Depalmitoylation Activity.

Dynamic post-translational modifications allow the rapid, specific, and tunable regulation of protein functions in eukaryotic cells. S-acylation is the only reversible lipid modification of proteins, in which a fatty acid, usually palmitate, is covalently attached to a cysteine residue of a protein by a zDHHC palmitoyl acyltransferase enzyme. Depalmitoylation is required for acylation homeostasis and is catalyzed by an enzyme from the alpha/beta hydrolase family of proteins usually acyl-protein thioesterase (APT1). The enzyme responsible for depalmitoylation in Trypanosoma brucei parasites is currently unknown. We demonstrate depalmitoylation activity in live bloodstream and procyclic form trypanosomes sensitive to dose-dependent inhibition with the depalmitoylation inhibitor, palmostatin B. We identified a homologue of human APT1 in Trypanosoma brucei which we named TbAPT-like (TbAPT-L). Epitope-tagging of TbAPT-L at N- and C- termini indicated a cytoplasmic localization. Knockdown or over-expression of TbAPT-L in bloodstream forms led to robust changes in TbAPT-L mRNA and protein expression but had no effect on parasite growth in vitro, or cellular depalmitoylation activity. Esterase activity in cell lysates was also unchanged when TbAPT-L was modulated. Unexpectedly, recombinant TbAPT-L possesses esterase activity with specificity for short- and medium-chain fatty acid substrates, leading to the conclusion, TbAPT-L is a lipase, not a depalmitoylase.

The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.

Formulation of a Thermosensitive Imaging Hydrogel for Topical Application and Rapid Visualization of Tumor Margins in the Surgical Cavity.

BACKGROUND: Tumor-positive surgical margins during primary breast cancer (BCa) surgery are associated with a two-fold increase in the risk of local recurrence when compared with tumor-negative margins. Pathological microscopic evaluation of the samples only assesses about 1/10 of 1% of the entire volume of the removed BCa specimens, leading to margin under-sampling and potential local recurrence in patients with pathologically clean margins, i.e., false negative margins. In the case of tumor-positive margins, patients need to undergo re-excision and/or radiation therapy, resulting in increases in complications, morbidity, and healthcare costs. Development of a simple real-time imaging technique to identify residual BCa in the surgical cavity rapidly and precisely could significantly improve the quality of care. METHODS: A small-molecule, fluorescently quenched protease-substrate probe, AKRO-QC-ICG, was tested as part of a thermosensitive imaging gel formulated for topical application and imaging of the BCa surgical cavity. RESULTS: More than forty formulations of gel mixtures were investigated to enable easy fluid application and subsequent solidification once applied, preventing dripping and pooling in the surgical cavity. The final formulation was tested using human BCa orthotopic implants in nude and NSG patient-derived xenografts (PDX) mice. This formulation of Pluronic F-127/DMSO/AKRO-QC-ICG imaging gel was found to be a good solvent for the probe, with a desirable thermo-reversible solid-gel transition and mechanical strength for distribution of AKRO-QC-ICG on the surfaces of tissue. It demonstrated excellent ability to detect BCa tissue after 10 min exposure, with a high signal-to-noise ratio both in mouse xenografts and freshly excised human lumpectomy tissue. The in vivo efficacy of the AKRO-QC-ICG imaging gel to detect BCa revealed the levels of sensitivity/specificity = 0.92/1 in 12 nude mice, which was corroborated with the sensitivity/specificity = 0.94/1 in 10 PDX mice. CONCLUSIONS: Utilization of Pluronic F-127/DMSO/AKRO-QC-ICG imaging gel for topical application to detect BCa in the surgical cavity during surgery has the potential to reduce re-excisions, with consequent savings in healthcare costs and enhancement in patient quality of life.

A Cathepsin-Targeted Quenched Activity-Based Probe Facilitates Enhanced Detection of Human Tumors during Resection.

PURPOSE: Fluorescence-guided surgery using tumor-targeted contrast agents has been developed to improve the completeness of oncologic resections. Quenched activity-based probes that fluoresce after covalently binding to tumor-specific enzymes have been proposed to improve specificity, but none have been tested in humans. Here, we report the successful clinical translation of a cathepsin activity-based probe (VGT-309) for fluorescence-guided surgery. EXPERIMENTAL DESIGN: We optimized the specificity, dosing, and timing of VGT-309 in preclinical models of lung cancer. To evaluate clinical feasibility, we conducted a canine study of VGT-309 during pulmonary tumor resection. We then conducted a randomized, double-blind, dose-escalation study in healthy human volunteers receiving VGT-309 to evaluate safety. Finally, we tested VGT-309 in humans undergoing lung cancer surgery. RESULTS: In preclinical models, we found highly specific tumor cell labeling that was blocked by a broad spectrum cathepsin inhibitor. When evaluating VGT-309 for guidance during resection of canine tumors, we found that the probe selectively labeled tumors and demonstrated high tumor-to-background ratio (TBR; range: 2.15-3.71). In the Phase I human study, we found that VGT-309 was safe at all doses studied. In the ongoing Phase II trial, we report two cases in which VGT-309 localized visually occult, non-palpable tumors (TBRs = 2.83 and 7.18) in real time to illustrate its successful clinical translation and potential to improve surgical management. CONCLUSIONS: This first-in-human study demonstrates the safety and feasibility of VGT-309 to label human pulmonary tumors during resection. These results may be generalizable to other cancers due to cathepsin overexpression in many solid tumors.

Integration of bioinformatic and chemoproteomic tools for the study of enzyme conservation in closely related bacterial species.

Activity-based protein profiling (ABPP) is a commonly utilized technique to globally characterize the endogenous activity of multiple enzymes within a related family. While it has been used extensively to identify enzymes that are differentially active across various mammalian tissues, recent efforts have expanded this technique to studying bacteria. As ABPP is applied to diverse sets of bacterial strains found in microbial communities, there is also an increasing need for robust tools for assessing the conservation of enzymes across closely related bacterial species and strains. In this chapter, we detail the integration of gel-based ABPP with basic bioinformatic tools to enable the analysis of enzyme activity, distribution, and homology. We use as an example the family of serine hydrolases identified in the skin commensal bacterium Staphylococcus epidermidis.

Activity-Based Diagnostics: Recent Advances in the Development of Probes for Use with Diverse Detection Modalities.

Abnormal enzyme expression and activity is a hallmark of many diseases. Activity-based diagnostics are a class of chemical probes that aim to leverage this dysregulated metabolic signature to produce a detectable signal specific to diseased tissue. In this Review, we highlight recent methodologies employed in activity-based diagnostics that provide exquisite signal sensitivity and specificity in complex biological systems for multiple disease states. We divide these examples based upon their unique signal readout modalities and highlight those that have advanced into clinical trials.

Identification of covalent inhibitors that disrupt M. tuberculosis growth by targeting multiple serine hydrolases involved in lipid metabolism.

The increasing incidence of antibiotic-resistant Mycobacterium tuberculosis infections is a global health threat necessitating the development of new antibiotics. Serine hydrolases (SHs) are a promising class of targets because of their importance for the synthesis of the mycobacterial cell envelope. We screen a library of small molecules containing serine-reactive electrophiles and identify narrow-spectrum inhibitors of M. tuberculosis growth. Using these lead molecules, we perform competitive activity-based protein profiling and identify multiple SH targets, including enzymes with uncharacterized functions. Lipidomic analyses of compound-treated cultures reveal an accumulation of free lipids and a substantial decrease in lipooligosaccharides, linking SH inhibition to defects in cell envelope biogenesis. Mutant analysis reveals a path to resistance via the synthesis of mycocerates, but not through mutations to SH targets. Our results suggest that simultaneous inhibition of multiple SH enzymes is likely to be an effective therapeutic strategy for the treatment of M. tuberculosis infections.