Cathepsin B promotes Aß proteotoxicity by modulating aging regulating mechanisms.

While the activities of certain proteases promote proteostasis and prevent neurodegeneration-associated phenotypes, the protease cathepsin B (CTSB) enhances proteotoxicity in Alzheimer's disease (AD) model mice, and its levels are elevated in brains of AD patients. How CTSB exacerbates the toxicity of the AD-causing Amyloid ß (Aß) peptide is controversial. Using an activity-based probe, aging-altering interventions and the nematode C. elegans, we discovered that the CTSB CPR-6 promotes Aß proteotoxicity but mitigates the toxicity of polyQ stretches. While the knockdown of cpr-6 does not affect lifespan, it alleviates Aß toxicity by reducing the expression of swsn-3 and elevating the level of the protein SMK-1, both involved in the regulation of aging. These observations unveil a mechanism by which CTSB aggravates Aß-mediated toxicity, indicate that it plays opposing roles in the face of distinct proteotoxic insults and highlight the importance of tailoring specific remedies for distinct neurodegenerative disorders.

Differential Effect of Simulated Microgravity on the Cellular Uptake of Small Molecules.

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes in membrane properties. SMG was applied to cultured cells using a random-positioning machine for up to three hours. We assessed the cellular accumulation of compounds representing substrates of uptake and efflux transporters, and of compounds not shown to be transported by membrane carriers. Exposure to SMG led to an increase of up to 60% (p < 0.01) in the cellular uptake of efflux transporter substrates, whereas a glucose transporter substrate showed a decrease of 20% (p < 0.05). The uptake of the cathepsin activity-based probe GB123 (MW, 1198 g/mol) was also enhanced (1.3-fold, p < 0.05). Cellular emission of molecules larger than ~3000 g/mol was reduced by up to 50% in SMG (p < 0.05). Our findings suggest that short-term exposure to SMG could differentially affect drug distribution across membranes. Longer exposure to microgravity, e.g., during spaceflight, may have distinct effects on the cellular uptake of small molecules.

Development and Application of Reversible and Irreversible Covalent Probes for Human and Mouse Cathepsin-K Activity Detection, Revealing Nuclear Activity.

Cathepsin-K (CTSK) is an osteoclast-secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity-based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro-form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards.

Real-time analysis of osteoclast resorption and fusion dynamics in response to bone resorption inhibitors.

Cathepsin K (CatK), an essential collagenase in osteoclasts (OCs), is a potential therapeutic target for the treatment of osteoporosis. Using live-cell imaging, we monitored the bone resorptive behaviour of OCs during dose-dependent inhibition of CatK by an ectosteric (Tanshinone IIA sulfonate) and an active site inhibitor (odanacatib). CatK inhibition caused drastic reductions in the overall resorption speed of OCs. At IC50 CatK-inhibitor concentration, OCs reduced about 40% of their trench-forming capacity and at fourfold IC50 concentrations, a > 95% reduction was observed. The majority of CatK-inhibited OCs (~ 75%) were involved in resorption-migration-resorption episodes forming adjacent pits, while ~ 25% were stagnating OCs which remained associated with the same excavation. We also observed fusions of OCs during the resorption process both in control and inhibitor-treated conditions, which increased their resorption speeds by 30-50%. Inhibitor IC50-concentrations increased OC-fusion by twofold. Nevertheless, more fusion could not counterweigh the overall loss of resorption activity by inhibitors. Using an activity-based probe, we demonstrated the presence of active CatK at the resorbing front in pits and trenches. In conclusion, our data document how OCs respond to CatK-inhibition with respect to movement, bone resorption activity, and their attempt to compensate for inhibition by activating fusion.

Challenges in Batch-to-Bed Translation Involving Inflammation-Targeting Compounds in Chronic Epilepsy: The Case of Cathepsin Activity-Based Probes.

Our goal was to test the feasibility of a new theranostic strategy in chronic epilepsy by targeting cathepsin function using novel cathepsin activity-based probes (ABPs). We assessed the biodistribution of fluorescent cathepsin ABPs in vivo, in vitro, and ex vivo, in rodents with pilocarpine-induced chronic epilepsy and naïve controls, in human epileptic tissue, and in the myeloid cell lines RAW 264.7 (monocytes) and BV2 (microglia). Distribution and localization of ABPs were studied by fluorescence scanning, immunoblotting, microscopy, and cross-section staining in anesthetized animals, in their harvested organs, in brain tissue slices, and in vitro. Blood-brain-barrier (BBB) efflux transport was evaluated in transporter-overexpressing MDCK cells and using an ATPase activation assay. Although the in vivo biodistribution of ABPs to both naïve and epileptic hippocampi was negligible, ex vivo ABPs bound cathepsins preferentially within epileptogenic brain tissue and colocalized with neuronal but not myeloid cell markers. Thus, our cathepsin ABPs are less likely to be of major clinical value in the diagnosis of chronic epilepsy, but they may prove to be of value in intraoperative settings and in CNS conditions with leakier BBB or higher cathepsin activity, such as status epilepticus.

Primer for Designing Main Protease (Mpro) Inhibitors of SARS-CoV-2.

The COVID-19 outbreak has been devastating, with hundreds of millions of infections and millions of deaths reported worldwide. In response, the application of structure-activity relationships (SAR) upon experimentally validated inhibitors of SARS-CoV-2 main protease (Mpro) may provide an avenue for the identification of new lead compounds active against COVID-19. Upon the basis of information gleaned from a combination of reported crystal structures and the docking of experimentally validated inhibitors, four "rules" for designing potent Mpro inhibitors have been proposed. The aim here is to guide medicinal chemists toward the most probable hits and to provide guidance on repurposing available structures as Mpro inhibitors. Experimental examination of our own previously reported inhibitors using the four "rules" identified a potential lead compound, the cathepsin inhibitor GB111-NH2, that was 2.3 times more potent than SARS-CoV-2 Mpro inhibitor N3.

Improved Cathepsin Probes for Sensitive Molecular Imaging.

Cysteine cathepsin proteases are found under normal conditions in the lysosomal compartments of cells, where they play pivotal roles in a variety of cellular processes such as protein and lipid metabolism, autophagy, antigen presentation, and cell growth and proliferation. As a consequence, aberrant localization and activity contribute to several pathologic conditions such as a variety of malignancies, cardiovascular diseases, osteoporosis, and other diseases. Hence, there is a resurgence of interest to expand the toolkit to monitor intracellular cathepsin activity and better ascertain their functions under these circumstances. Previous fluorescent activity-based probes (ABPs) that target cathepsins B, L, and S enabled detection of their activity in intact cells as well as non-invasive detection in animal disease models. However, their binding potency is suboptimal compared to the cathepsin inhibitor on which they were based, as the P1 positive charge was capped by a reporter tag. Here, we show the development of an improved cathepsin ABP that has a P1 positive charge by linking the tag on an additional amino acid at the end of the probe. While enhancing potency towards recombinant cathepsins, the new probe had reduced cell permeability due to additional peptide bonds. At a second phase, the probe was trimmed; the fluorophore was linked to an extended carbobenzoxy moiety, leading to enhanced cell permeability and superb detection of cathepsin activity in intact cells. In conclusion, this work introduces a prototype design for the next generation of highly sensitive ABPs that have excellent detection of cellular cathepsin activity.

Determination of potential inhibitors based on isatin derivatives against SARS-CoV-2 main protease (mpro): a molecular docking, molecular dynamics and structure-activity relationship studies.

SARS-COV-2, the novel coronavirus and root of global pandemic COVID-19 caused a severe health threat throughout the world. Lack of specific treatments raised an effort to find potential inhibitors for the viral proteins. The recently invented crystal structure of SARS-CoV-2 main protease (Mpro) and its key role in viral replication; non-resemblance to any human protease makes it a perfect target for inhibitor research. This article reports a computer-aided drug design (CADD) approach for the screening of 118 compounds with 16 distinct heterocyclic moieties in comparison with 5 natural products and 7 repurposed drugs. Molecular docking analysis against Mpro protein were performed finding isatin linked with a oxidiazoles (A2 and A4) derivatives to have the best docking scores of -11.22 kcal/mol and -11.15 kcal/mol respectively. Structure-activity relationship studies showed a good comparison with a known active Mpro inhibitor and repurposed drug ebselen with an IC50 value of -0.67 µM. Molecular Dynamics (MD) simulations for 50 ns were performed for A2 and A4 supporting the stability of the two compounds within the binding pocket, largely at the S1, S2 and S4 domains with high binding energy suggesting their suitability as potential inhibitors of Mpro for SARS-CoV-2.

Kobophenol A Inhibits Binding of Host ACE2 Receptor with Spike RBD Domain of SARS-CoV-2, a Lead Compound for Blocking COVID-19.

In the search for inhibitors of COVID-19, we have targeted the interaction between the human angiotensin-converting enzyme 2 (ACE2) receptor and the spike receptor binding domain (S1-RBD) of SARS-CoV-2. Virtual screening of a library of natural compounds identified Kobophenol A as a potential inhibitor. Kobophenol A was then found to block the interaction between the ACE2 receptor and S1-RBD in vitro with an IC50 of 1.81 ± 0.04 µM and inhibit SARS-CoV-2 viral infection in cells with an EC50 of 71.6 µM. Blind docking calculations identified two potential binding sites, and molecular dynamics simulations predicted binding free energies of -19.0 ± 4.3 and -24.9 ± 6.9 kcal/mol for Kobophenol A to the spike/ACE2 interface and the ACE2 hydrophobic pocket, respectively. In summary, Kobophenol A, identified through docking studies, is the first compound that inhibits SARS-CoV-2 binding to cells through blocking S1-RBD to the host ACE2 receptor and thus may serve as a good lead compound against COVID-19.

TNFα expression by Porphyromonas gingivalis-stimulated macrophages relies on Sirt1 cleavage.

OBJECTIVE: Periodontitis is one the most common chronic inflammatory conditions, resulting in destruction of tooth-supporting tissues and leading to tooth loss. Porphyromonas gingivalis activates host macrophages to secrete pro-inflammatory cytokines and elicit tissue damage, in part by inducing NF-kappa-B transactivation. Since NFκB transactivation is negatively regulated by the Nicotinamide adenine dinucleotide (NAD)-dependent deacetylase enzyme Sirt1, we sought to assess if RAW264.7 macrophages exposed to P. gingivalis demonstrate impaired Sirt1 activity, to ultimately induce a pro-inflammatory response. METHODS: RAW264.7 macrophages were incubated with heat- killed P. gingivalis for 2, 4, 8, and 24 h. Stimulated RAW264.7 were assessed for TNFα expression via PCR, ELISA, and ChIP analysis. Following the activation of RAW264.7 macrophages, immunoblot analysis was executed to detect modifications in Sirt1 and the NFκB subunit RelA that is essential for NFκB transcriptional activity. RESULTS: TNFα expression was elevated 4 h after exposure to P. gingivalis. ChIP confirmed that RelA was enriched in the mouse TNFα promoter 4 h following stimulation, which correlated with the increased TNFα mRNA levels. Preceding TNFα expression, we detected Phosphoserine 536 and acetylated lysine 310 of RelA after 2 hours exposure with P. gingivalis. Moreover, reduced Sirt1 activity was associated with its cleavage in RAW264.7 protein extracts, after 2 hours of P. gingivalis exposure. Blocking TLR2/4 signaling prevented Sirt1 cleavage, loss of deacetylase activity, and TNFα secretion, while co-administering CA074Me (a cathepsin B inhibitor) with P. gingivalis reduced RelA promoter enrichment, resulting in impaired TNFα expression. CONCLUSIONS: Together, the results suggest that P. gingivalis induces TNFα expression, at least in part, by enhancing cleavage of Sirt1 via a TLR-dependent signaling circuit.

IRS1 phosphorylation underlies the non-stochastic probability of cancer cells to persist during EGFR inhibition therapy.

Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment and has a unimodal distribution ranging from 0 to almost 100%. Notably, CTP is drug specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under epidermal growth factor receptor inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-epidermal growth factor receptor therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using new rationally designed drug combinations.

Cathepsin Inhibition Modulates Metabolism and Polarization of Tumor-Associated Macrophages.

Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH2, led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE2, which correlated with the upregulation of numerous NFkB-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu.

Cathepsins Drive Anti-Inflammatory Activity by Regulating Autophagy and Mitochondrial Dynamics in Macrophage Foam Cells.

BACKGROUND/AIMS: Atherosclerosis underlies the majority of cardiovascular events, consequent to non-resolving inflammation. Considerable evidence implicates autophagy dysfunction at the core of this inflammatory condition, but the basis of this dysfunction is not fully understood. METHODS: Using an in vitro model of lipid-laden macrophages, activity-based probes and high-throughput techniques, we studied the role of the cysteine proteases cathepsins in autophagy. RESULTS: We showed that cathepsin activity is suppressed by oxidized lipids and that cathepsin has an indispensable role in the autophagy-lysosomal degradation pathway. Accordingly, loss of cathepsin function resulted in autophagy derangement. Shotgun proteomics confirmed autophagy dysfunction and unveiled a pivotal role of cathepsin L in a putative cathepsin degradation network. At the physiological level, cathepsin inhibition resulted in mitochondrial stress, which translated into impaired oxidative metabolism, excessive production of reactive oxygen species and activation of the cellular stress response, driven by ATF4-CHOP transcription factors. In addition, transcriptomic analysis of these cells uncovered some genetic similarities with the inflammatory macrophage phenotype (a.k.a M1 macrophages) and increased expression of inflammatory cytokines. CONCLUSION: Our data highlight the importance of cathepsins for mitochondrial quality control mechanisms and amelioration of vascular inflammation.

A Theranostic Cathepsin Activity-Based Probe for Noninvasive Intervention in Cardiovascular Diseases.

Despite the common use of lipid-lowering medications, cardiovascular diseases continue to be a significant health concern. Atherosclerosis, one of the most frequent causes of cardiovascular morbidity, involves extensive inflammatory activity and remodeling of the vascular endothelium. This relentless inflammatory condition can ultimately give rise to clinical manifestations, such as ischemic heart disease or stroke. Accumulating evidence over the past decades implicates cysteine protease cathepsins in cardiovascular disorders. In particular, Cathepsins B, L, and S are over-expressed during vascular inflammation, and their activity is associated with impaired clinical outcomes. Here we took advantage of these molecular events to introduce a non-invasive detection and treatment approach to modulate vascular inflammation using a Photosensitizing quenched Activity-Based Probed (PS-qABP) that targets these proteases. Methods: We tested the application of this approach in LDL receptor-deficient mice and used non-invasive imaging and heart cross-section staining to assess the theranostic efficacy of this probe. Moreover, we used fresh human endarterectomy tissues to analyze cathepsin signals on gel, and verified cathepsin identity by mass spectrometry. Results: We showed that our PS-qABP can rapidly accumulate in areas of inflammatory atheromas in vivo, and application of light therapy profoundly reduced lesional immune cell content without affecting smooth muscle cell and collagen contents. Lastly, using human tissue samples we provided proof-of-concept for future clinical applications of this technology. Conclusions: Photodynamic therapy guided by cysteine cathepsin activity is an effective approach to reduce vascular inflammation and attenuate atherosclerosis progression. This approach could potentially be applied in clinical settings.

Cathepsin L Regulates Metabolic Networks Controlling Rapid Cell Growth and Proliferation.

Rapidly proliferating cells reshape their metabolism to satisfy their ever-lasting need for cellular building blocks. This phenomenon is exemplified in certain malignant conditions such as cancer but also during embryonic development when cells rely heavily on glycolytic metabolism to exploit its metabolic intermediates for biosynthetic processes. How cells reshape their metabolism is not fully understood. Here we report that loss of cathepsin L (Cts L) is associated with a fast proliferation rate and enhanced glycolytic metabolism that depend on lactate dehydrogenase A (LDHA) activity. Using mass spectrometry analysis of cells treated with a pan cathepsin inhibitor, we observed an increased abundance of proteins involved in central carbon metabolism. Further inspection of putative Cts L targets revealed an enrichment for glycolytic metabolism that was independently confirmed by metabolomic and biochemical analyses. Moreover, proteomic analysis of Cts L-knockout cells identified LDHA overexpression that was demonstrated to be a key metabolic junction in these cells. Lastly, we show that Cts L inhibition led to increased LDHA protein expression, suggesting a causal relationship between LDHA expression and function. In conclusion, we propose that Cts L regulates this metabolic circuit to keep cell division under control, suggesting the therapeutic potential of targeting this protein and its networks in cancer.

CT Imaging of Enzymatic Activity in Cancer Using Covalent Probes Reveal a Size-Dependent Pattern.

X-ray CT instruments are among the most available, efficient, and cost-effective imaging modalities in hospitals. The field of CT molecular imaging is emerging which relies mainly on the detection of gold nanoparticles and iodine-containing compounds directed to tagging a variety of abundant biomolecules. Here for the first time we attempted to detect enzymatic activity, while the low sensitivity of CT scanners to contrast reagents made this a challenging task. Therefore, we developed a new class of nanosized cathepsin-targeted activity-based probes (ABPs) for functional CT imaging of cancer. ABPs are small molecules designed to covalently modify enzyme targets in an activity-dependent manner. Using a CT instrument, these novel probes enable detection of the elevated cathepsin activity within cancerous tissue, thus creating a direct link between biological processes and imaging signals. We present the generation and biochemical evaluation of a library of ABPs tagged with different sized gold nanoparticles (GNPs), with various ratios of cathepsin-targeting moiety and a combination of different polyethylene glycol (PEG) protective layers. The most potent and stable GNP-ABPs were applied for noninvasive cancer imaging in mice. Surprisingly, detection of CT contrast from the tumor had reverse correlation to GNP size and the amount of targeting moiety. Interestingly, TEM images of tumor sections show intercellular lysosomal subcellular localization of the GNP-ABPs. In conclusion, we demonstrate that the covalent linkage is key for detection using low sensitive imaging modalities and the utility of GNP-ABPs as a promising tool for enzymatic-based CT imaging.

mTORC1 activation in B cells confers impairment of marginal zone microarchitecture by exaggerating cathepsin activity.

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood-borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1BKO ) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underlying mechanisms for MZ B-cell loss, we have analysed the spleen MZ architecture of TSC1BKO mice and found it to be severely impaired. Examination of lymphotoxins (LTα and LTß) and lymphotoxin receptor (LTßR) expression indicated that LTßR levels in spleen stroma were reduced by TSC1 deletion in the B cells. Furthermore, LTα transcripts in B cells were reduced. Because LTßR is sensitive to proteolysis, we analysed cathepsin activity in TSC1BKO . A higher cathepsin activity, particularly of cathepsin B, was observed, which was reduced by mTORC1 inhibition with rapamycin in vivo. Remarkably, in vivo administration of a pan-cathepsin inhibitor restored LTßR expression, LTα mRNA levels and the MZ architecture. Our data identify a novel connection, although not elucidated at the molecular level, between mTORC1 and cathepsin activity in a manner relevant to MZ dynamics.

Image-guided surgery using near-infrared Turn-ON fluorescent nanoprobes for precise detection of tumor margins.

Complete tumor removal during surgery has a great impact on patient survival. To that end, the surgeon should detect the tumor, remove it and validate that there are no residual cancer cells left behind. Residual cells at the incision margin of the tissue removed during surgery are associated with tumor recurrence and poor prognosis for the patient. In order to remove the tumor tissue completely with minimal collateral damage to healthy tissue, there is a need for diagnostic tools that will differentiate between the tumor and its normal surroundings. Methods: We designed, synthesized and characterized three novel polymeric Turn-ON probes that will be activated at the tumor site by cysteine cathepsins that are highly expressed in multiple tumor types. Utilizing orthotopic breast cancer and melanoma models, which spontaneously metastasize to the brain, we studied the kinetics of our polymeric Turn-ON nano-probes. Results: To date, numerous low molecular weight cathepsin-sensitive substrates have been reported, however, most of them suffer from rapid clearance and reduced signal shortly after administration. Here, we show an improved tumor-to-background ratio upon activation of our Turn-ON probes by cathepsins. The signal obtained from the tumor was stable and delineated the tumor boundaries during the whole surgical procedure, enabling accurate resection. Conclusions: Our findings show that the control groups of tumor-bearing mice, which underwent either standard surgery under white light only or under the fluorescence guidance of the commercially-available imaging agents ProSense® 680 or 5-aminolevulinic acid (5-ALA), survived for less time and suffered from tumor recurrence earlier than the group that underwent image-guided surgery (IGS) using our Turn-ON probes. Our "smart" polymeric probes can potentially assist surgeons' decision in real-time during surgery regarding the tumor margins needed to be removed, leading to improved patient outcome.

Molecular Imaging of Cancer Using X-ray Computed Tomography with Protease Targeted Iodinated Activity-Based Probes.

X-ray computed tomography (CT) is a robust, precise, fast, and reliable imaging method that enables excellent spatial resolution and quantification of contrast agents throughout the body. However, CT is largely inadequate for molecular imaging applications due mainly to its low contrast sensitivity that forces the use of large concentrations of contrast agents for detection. To overcome this limitation, we generated a new class of iodinated nanoscale activity-based probes (IN-ABPs) that sufficiently accumulates at the target site by covalently binding cysteine cathepsins that are exceptionally highly expressed in cancer. The IN-ABPs are comprised of a short targeting peptide selective to specific cathepsins, an electrophilic moiety that allows activity-dependent covalent binding, and tags containing dendrimers with up to 48 iodine atoms. IN-ABPs selectively bind and inhibit activity of recombinant and intracellular cathepsin B, L, and S. We compared the in vivo kinetics, biodistribution, and tumor accumulation of IN-ABPs bearing 18 and 48 iodine atoms each, and their control counterparts lacking the targeting moiety. Here we show that although both IN-ABPs bind specifically to cathepsins within the tumor and produce detectable CT contrast, the 48-iodine bearing IN-ABP was found to be optimal with signals over 2.1-fold higher than its nontargeted counterpart. In conclusion, this study shows the synthetic feasibility and potential utility of IN-ABPs as potent contrast agents that enable molecular imaging of tumors using CT.