Pharmacological Polarization of Tumor-Associated Macrophages Toward a CXCL9 Antitumor Phenotype.

Tumor-associated macrophages (TAM) are a diverse population of myeloid cells that are often abundant and immunosuppressive in human cancers. CXCL9Hi TAM has recently been described to have an antitumor phenotype and is linked to immune checkpoint response. Despite the emerging understanding of the unique antitumor TAM phenotype, there is a lack of TAM-specific therapeutics to exploit this new biological understanding. Here, the discovery and characterization of multiple small-molecule enhancers of chemokine ligand 9 (CXCL9) and their targeted delivery in a TAM-avid systemic nanoformulation is reported. With this strategy, it is efficient encapsulation and release of multiple drug loads that can efficiently induce CXCL9 expression in macrophages, both in vitro and in vivo in a mouse tumor model. These observations provide a window into the molecular features that define TAM-specific states, an insight a novel therapeutic anticancer approach is used to discover.

A Universal and Modular Scaffold for Heparanase Activatable Probes and Drugs.

Heparanase (HPSE) is an endo-ß-glucuronidase involved in extracellular matrix remodeling in rapidly healing tissues, most cancers and inflammation, and viral infection. Its importance as a therapeutic target warrants further study, but such is hampered by a lack of research tools. To expand the toolkits for probing HPSE enzymatic activity, we report the design of a substrate scaffold for HPSE comprised of a disaccharide substrate appended with a linker, capable of carrying cargo until being cleaved by HPSE. Here exemplified as a fluorogenic, coumarin-based imaging probe, this scaffold can potentially expand the availability of HPSE-responsive imaging or drug delivery tools using a variety of imaging moieties or other cargo. We show that electronic tuning of the scaffold provides a robust response to HPSE while simplifying the structural requirements of the attached cargo. Molecular docking and modeling suggest a productive probe/HPSE binding mode. These results further support the hypothesis that the reactivity of these HPSE-responsive probes is predominantly influenced by the electron density of the aglycone. This universal HPSE-activatable scaffold will greatly facilitate future development of HPSE-responsive probes and drugs.

Molecular probes for selective detection of cysteine cathepsins.

Cysteine cathepsins are proteases critical in physiopathological processes and show potential as targets or biomarkers for diseases and medical conditions. The 11 members of the cathepsin family are redundant in some cases but remarkably independent of others, demanding the development of both pan-cathepsin targeting tools as well as probes that are selective for specific cathepsins with little off-target activity. This review addresses the diverse design strategies that have been employed to accomplish this tailored selectivity among cysteine cathepsin targets and the imaging modalities incorporated. The power of these diverse tools is contextualized by briefly highlighting the nature of a few prominent cysteine cathepsins, their involvement in select diseases, and the application of cathepsin imaging probes in research spanning basic biochemical studies to clinical applications.

Dual-Mechanism Quenched Fluorogenic Probe Provides Selective and Rapid Detection of Cathepsin L Activity*.

Cathepsin L (CTL) is a cysteine protease demonstrating upregulated activity in many disease states. Overlapping substrate specificity makes selective detection of CTL activity difficult to parse from that of its close homologue CTV and the ubiquitous CTB. Current probes of CTL activity have limited applications due to either poor contrast or extra assay steps required to achieve selectivity. We have developed a fluorogenic probe, CTLAP, that displays good selectivity for CTL over CTB and CTV while exhibiting low background fluorescence attributed to dual quenching mechanisms. CTLAP achieves optimum CTL selectivity in the first 10 min of incubation, thus suggesting that it is amenable for rapid detection of CTL, even in the presence of competing cathepsins.

Responsive Fluorophore Aggregation Provides Spectral Contrast for Fluorescence Lifetime Imaging.

Fluorophores experience altered emission lifetimes when incorporated into and liberated from macromolecules or molecular aggregates; this trend suggests the potential for a fluorescent, responsive probe capable of undergoing self-assembly and aggregation and consequently altering the lifetime of its fluorescent moiety to provide contrast between the active and inactive probes. We developed a cyanobenzothioazole-fluorescein conjugate (1), and spectroscopically examined the lifetime changes caused by its reduction-induced aggregation in vitro. A decrease in lifetime was observed for compound 1 in a buffered system activated by the biological reducing agent glutathione, thus suggesting a possible approach for designing responsive self-aggregating lifetime imaging probes.

Ultrasensitive small molecule fluorogenic probe for human heparanase.

Heparanase (HPA) is a critical enzyme involved in the remodeling of the extracellular matrix (ECM), and its elevated expression has been linked with diseases such as various types of cancer and inflammation. The detection of heparanase enzymatic activity holds tremendous value in the study of the cellular microenvironment, and search of molecular therapeutics targeting heparanase, however, no structurally defined probes are available for the detection of heparanase activity. Here we present the development of the first ultrasensitive fluorogenic small-molecule probe for heparanase enzymatic activity via tuning the electronic effect of the substrate. The probe exhibits a 756-fold fluorescence turn-on response in the presence of human heparanase, allowing one-step detection of heparanase activity in real-time with a picomolar detection limit. The high sensitivity and robustness of the probe are exemplified in a high-throughput screening assay for heparanase inhibitors.