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.

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.

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.

A synthetic covalent ligand of the C/EBPß transactivation domain inhibits acute myeloid leukemia cells.

C/EBPß has recently emerged as a pro-leukemogenic transcription factor that cooperates with oncoprotein MYB to maintain proliferation and differentiation block of AML cells, making C/EBPß an interesting drug target for AML. Here we have studied the inhibitory potential and biological effects of a synthetic analog of the natural product helenalin, a known inhibitor of C/EBPß. The synthetic compound inhibits C/EBPß by covalent binding to cysteine residues in the transactivation domain, thereby causing up-regulation of differentiation-associated genes, cell death and reduced self-renewal potential of AML cells. Suppression of these effects by ectopic expression of C/EBPß or MYB and gene expression profiling validate C/EBPß as a relevant target of the helenalin-mimic and highlight its role as a pro-leukemogenic factor. Overall, our work demonstrates that the synthetic helenalin mimic acts as a covalent inhibitor of C/EBPß and identifies the cysteine residues in the transactivation domain of C/EBPß as ligandable sites. The helenalin mimic can be considered a potential "lead molecule" but needs further development towards more effective C/EBPß inhibitors before being used as a therapeutic agent.

Challenges for Targeting SARS-CoV-2 Proteases as a Therapeutic Strategy for COVID-19.

Two proteases produced by the SARS-CoV-2 virus, the main protease and papain-like protease, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsins L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

AND-gate contrast agents for enhanced fluorescence-guided surgery.

Surgical resection of tumours requires precisely locating and defining the margins between lesions and normal tissue. However, this is made difficult by irregular margin borders. Although molecularly targeted optical contrast agents can be used to define tumour margins during surgery in real time, the selectivity of the contrast agents is often limited by the target being expressed in both healthy and tumour tissues. Here, we show that AND-gate optical imaging probes that require the processing of two substrates by multiple tumour-specific enzymes produce a fluorescent signal with significantly improved specificity and sensitivity to tumour tissue. We evaluated the performance of the probes in mouse models of mammary tumours and of metastatic lung cancer, as well as during fluorescence-guided robotic surgery. Imaging probes that rely on multivariate activation to selectively target complex patterns of enzymatic activity should be useful in disease detection, treatment and monitoring.

Synthesis of Guaianolide Analogues with a Tunable α-Methylene-γ-lactam Electrophile and Correlating Bioactivity with Thiol Reactivity.

α-Methylene-γ-lactones are present in ∼3% of known natural products, and compounds comprising this motif display a range of biological activities. However, this reactive lactone limits informed structure-activity relationships for these bioactive molecules. Herein, we describe chemically tuning the electrophilicity of the α-methylene-γ-lactone by replacement with an α-methylene-γ-lactam. Guaianolide analogues having α-methylene-γ-lactams are synthesized using the allenic Pauson-Khand reaction. Substitution of the lactam nitrogen with electronically different groups affords diverse thiol reactivity. Cellular NF-κB inhibition assays for these lactams were benchmarked against parthenolide and a synthetic α-methylene-γ-lactone showing a positive correlation between thiol reactivity and bioactivity. Cytotoxicity assays show good correlation at the outer limits of thiol reactivity but less so for compounds with intermediate reactivity. A La assay to detect reactive molecules by nuclear magnetic resonance and mass spectrometry peptide sequencing assays with the La antigen protein demonstrate that lactam analogues with muted nonspecific thiol reactivities constitute a better electrophile for rational chemical probe and therapeutic molecule design.

Methods for analysis of near-infrared (NIR) quenched-fluorescent contrast agents in mouse models of cancer.

Optical contrast agents containing near-infrared (NIR) fluorophores are useful for visualizing biological landmarks, enzyme activities and biological processes in live animals and humans. Activatable (smart) quenched-fluorescent probes are sensors that become fluorescent after processing by an enzyme or in response to a physiological change (i.e., pH, ROS, etc.). Recently, there has been increased interest in developing activatable probes for research and clinical applications. This requires evaluation using in vivo animal models to gain insights into the pharmacodynamic and pharmacokinetic properties of a given probe. Important parameters to measure when evaluating quenched-fluorescent probes are signal brightness and signal-to-background ratios, which define the sensitivity and specificity of a probe. In this chapter, we discuss methods to evaluate activatable quenched-fluorescent probes in mouse models of cancer. Quantification of fluorescent signal intensity, calculation of tumor-to-background ratios, comparison of fluorescent activation in specific organ compartments, and fluorescence scanning of sectioned tissue will be discussed.

Molecular Basis for the N-Terminal Bromodomain-and-Extra-Terminal-Family Selectivity of a Dual Kinase-Bromodomain Inhibitor.

As regulators of transcription, epigenetic proteins that interpret post-translational modifications to N-terminal histone tails are essential for maintaining cellular homeostasis. When dysregulated, "reader" proteins become drivers of disease. In the case of bromodomains, which recognize N-ε-acetylated lysine, selective inhibition of individual bromodomain-and-extra-terminal (BET)-family bromodomains has proven challenging. We describe the >55-fold N-terminal-BET bromodomain selectivity of 1,4,5-trisubstituted-imidazole dual kinase-bromodomain inhibitors. Selectivity for the BRD4 N-terminal bromodomain (BRD4(1)) over its second bromodomain (BRD4(2)) arises from the displacement of ordered waters and the conformational flexibility of lysine-141 in BRD4(1). Cellular efficacy was demonstrated via reduction of c-Myc expression, inhibition of NF-κB signaling, and suppression of IL-8 production through potential synergistic inhibition of BRD4(1) and p38α. These dual inhibitors provide a new scaffold for domain-selective inhibition of BRD4, the aberrant function of which plays a key role in cancer and inflammatory signaling.

SN-38 Conjugated Gold Nanoparticles Activated by Ewing Sarcoma Specific mRNAs Exhibit In Vitro and In Vivo Efficacy.

The limited delivery of chemotherapy agents to cancer cells and the nonspecific action of these agents are significant challenges in oncology. We have previously developed a customizable drug delivery and activation system in which a nucleic acid functionalized gold nanoparticle (Au-NP) delivers a drug that is selectively activated within a cancer cell by the presence of an mRNA unique to the cancer cell. The amount of drug released from sequestration to the Au-NP is determined by both the presence and the abundance of the cancer cell specific mRNA in a cell. We have now developed this technology for the potent, but difficult to deliver, topoisomerase I inhibitor SN-38. Herein, we demonstrate both the efficient delivery and selective release of SN-38 from gold nanoparticles in Ewing sarcoma cells with resulting efficacy in vitro and in vivo. These results provide further preclinical validation for this novel cancer therapy and may be extendable to other cancers that exhibit sensitivity to topoisomerase I inhibitors.

Helenalin Analogues Targeting NF-κB p65: Thiol Reactivity and Cellular Potency Studies of Varied Electrophiles.

Helenalin is a pseudoguaianolide natural product that targets Cys38 within the DNA binding domain of NF-κB transcription factor p65 (RelA). Helenalin contains two Michael acceptors that covalently modify cysteines: a α-methylene-γ-butyrolactone and a cyclopentenone. We recently reported two simplified helenalin analogues that mimic the biological activity of helenalin and contain both electrophilic moieties. To determine the individual contributions of the Michael acceptors toward NF-κB inhibition, we synthesized a small library of helenalin-based analogues containing various combinations of α-methylene-γ-butyrolactones and cyclopentenones. The kinetics of thiol addition to a subset of the analogues was measured to determine the relative thiol reactivities of the embedded electrophiles. Additionally, the cellular NF-κB inhibitory activities of the analogues were determined to elucidate the contributions of each Michael acceptor to biological potency. Our studies suggest the α-methylene-γ-butyrolactone contributes most significantly to the NF-κB inhibition of our simplified helenalin analogues.

Targeting NF-κB p65 with a Helenalin Inspired Bis-electrophile.

The canonical NF-κB signaling pathway is a mediator of the cellular inflammatory response and a target for developing therapeutics for multiple human diseases. The furthest downstream proteins in the pathway, the p50/p65 transcription factor heterodimer, have been recalcitrant toward small molecule inhibition despite the substantial number of compounds known to inhibit upstream proteins in the activation pathway. Given the roles of many of these upstream proteins in multiple biochemical pathways, targeting the p50/p65 heterodimer offers an opportunity for enhanced on-target specificity. Toward this end, the p65 protein presents two nondisulfide cysteines, Cys38 and Cys120, at its DNA-binding interface that are amenable to targeting by covalent molecules. The natural product helenalin, a sesquiterpene lactone, has been previously shown to target Cys38 on p65 and ablate its DNA-binding ability. Using helenalin as inspiration, simplified helenalin analogues were designed, synthesized, and shown to inhibit induced canonical NF-κB signaling in cell culture. Moreover, two simplified helenalin probes were proficient at forming covalent protein adducts, binding to Cys38 on recombinant p65, and targeting p65 in HeLa cells without engaging canonical NF-κB signaling proteins IκBα, p50, and IKKα/ß. These studies further support that targeting the p65 transcription factor-DNA interface with covalent small molecule inhibitors is a viable approach toward regulating canonical NF-κB signaling.

Covalent Modifiers: A Chemical Perspective on the Reactivity of α,ß-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions.

Although Michael acceptors display a potent and broad spectrum of bioactivity, they have largely been ignored in drug discovery because of their presumed indiscriminate reactivity. As such, a dearth of information exists relevant to the thiol reactivity of natural products and their analogues possessing this moiety. In the midst of recently approved acrylamide-containing drugs, it is clear that a good understanding of the hetero-Michael addition reaction and the relative reactivities of biological thiols with Michael acceptors under physiological conditions is needed for the design and use of these compounds as biological tools and potential therapeutics. This Perspective provides information that will contribute to this understanding, such as kinetics of thiol addition reactions, bioactivities, as well as steric and electronic factors that influence the electrophilicity and reversibility of Michael acceptors. This Perspective is focused on α,ß-unsaturated carbonyls given their preponderance in bioactive natural products.

Alkyne Ligation Handles: Propargylation of Hydroxyl, Sulfhydryl, Amino, and Carboxyl Groups via the Nicholas Reaction.

The Nicholas reaction has been applied to the installation of alkyne ligation handles. Acid-promoted propargylation of hydroxyl, sulfhydryl, amino, and carboxyl groups using dicobalt hexacarbonyl-stabilized propargylium ions is reported. This method is useful for introduction of propargyl groups into base-sensitive molecules, thereby expanding the toolbox of methods for the incorporation of alkynes for bio-orthogonal reactions. High-value molecules are used as the limiting reagent, and various propargylium ion precursors are compared.

Synthesis and antileukemic activities of C1-C10-modified parthenolide analogues.

Parthenolide (PTL) is a sesquiterpene lactone natural product with anti-proliferative activity to cancer cells. Selective eradication of leukemic stem cells (LSCs) over healthy hematopoietic stem cells (HSCs) by PTL has been demonstrated in previous studies, which suggests PTL and related molecules may be useful for targeting LSCs. Eradication of LSCs is required for curative therapy. Chemical optimizations of PTL to improve potency and pharmacokinetic parameters have focused largely on the α-methylene-γ-butyrolactone, which is essential for activity. Conversely, we evaluated modifications to the C1-C10 olefin and benchmarked new inhibitors to PTL with respect to inhibitory potency across a panel of cancer cell lines, ability to target drug-resistant acute myeloid leukemia (AML) cells, efficacy for inhibiting clonal growth of AML cells, toxicity to healthy bone marrow cells, and efficiency for promoting intracellular reactive oxygen species (ROS) levels. Cyclopropane 4 was found to possess less toxicity to healthy bone marrow cells, enhanced potency for the induction of cellular ROS, and similar broad-spectrum anti-proliferative activity to cancer cells in comparison to PTL.

A redox economical synthesis of bioactive 6,12-guaianolides.

Syntheses of two 6,12-guaianolide analogs are reported within. The scope of the tandem allylboration/lactonization chemistry is expanded to provide a functionalized allene-yne-containing α-methylene butyrolactone that undergoes a Rh(I)-catalyzed cyclocarbonylation reaction to afford a 5-7-5 ring system. The resulting cycloadducts bear a structural resemblance to other NF-κB inhibitors such as cumambrin A and indeed were shown to inhibit NF-κB signaling and cancer cell growth.