Beyond basic research: the contribution of cathepsin B to cancer development, diagnosis and therapy

Figures & data

Figure 1. The protein expression, trafficking, and targeted detection of cathepsin B (CtsB). Transcribed CtsB is translated and enters the secretory pathway and upon glycosylation resides in perinuclear lysosomes. Under specific cell stimulatory conditions or upon overexpression, CtsB can translocate to peripheral lysosomes and is secreted, where it can modulate extracellular matrix dynamics. Alternatively, CtsB can leak from lysosomes, process apoptotic proteins such as Bid and BAX which contribute to mitochondrial outer membrane permeabilization (MOMP) and apoptosis. CtsB can also reside in the nucleus of some cells as a result of it leaking from lysosomes as a mature protease, or after being expressed as a non-secreted CtsB isoform protein. Quench fluorescence Activity-Based probes (qABP), Photo-Sensitized ABPs (PS-ABP), or Prodrugs have been designed and tested to detect or target compartmentalized mature CtsB within the Extracellular Matrix (ECM) or within the intracellular endo-lysosomal compartments.

Figure 2. Utilization of Cathepsin B (CtsB) detection or activity in cancer diagnostic assays or therapy. Activated-Based Probes (ABP), designed with a warhead moiety (read arrowhead), a CtsB recognition/cleavage sequence (such as GFLG, white box), and a fluorescent excitable probe (green circle) allow CtsB detection. The fluorescent tag can also be quenched by an accompanying chemical moiety, as in qABP substrates (pale green circle). PS-qABPs are composed of an excitation-detectable fluorescent tag (green circle) and a specific chemical intermediate (yellow circle), which inflicts cytotoxic effects can be released upon photosensitization. Fluorescent Prodrugs contain a CtsB cleavage site, which determines the release of an active drug (yellow circle) and a fluorescent tag for detection purposes (green circle).

Table 1. Activity-Based Probes (ABPs) specific for detecting cathepsin proteases are highlighted. ABPs were tested for specific cathepsin protease detection using Gel-Labeling assays, cellular imaging approaches or in whole animal systems. The diseases that the ABPs have been tested for are also highlighted. NA-Not Applicable; Y-Yes; Mac-Macrophages; OA-Osteoarthritis; BC-Breast Cancer; K-Kidney; M-Mouse; AS-Atherosclerosis; KidC-Kidney Cancer.

ABP probe	Target Cathepsin	Warhead	Gel Labelling	Cell Imaging	In vivo Testing	Disease	Article
GB123	All Proteases and B, L, S	Acyloxymethyl ketone	NA	NA	M	BC	[16,152]
GB138	All Proteases and B, L	Acyloxymethyl ketone	Y	NA	M	BC	[152]
GB123	All Proteases and B, L, S	Acyloxymethyl ketone	NA	Mac	M	AS	[16,153]
GB123	B, S	Acyloxymethyl ketone	Y	OA	NA	OA	[154]
KDA-1	B	Fumarate diamine	Y	BC, K	NA	BC, KidC	[155]

Table 2. Quenched Activity-Based Probes (qABPs) specific for detecting cathepsin proteases are highlighted. qABPs were tested for specific cathepsin protease detection using Gel-Labeling assays, cellular imaging approaches or in whole animal systems. The diseases that the qABPs have been tested for are also highlighted. NA-Not Applicable; Y-Yes; BMM-Bone Marrow Macrophages; Mac-Macrophages; M-Mouse; BC-Breast Cancer; AS-Atherosclerosis; PrC-Prostate Cancer; CerC-Cervical Cancer; KidC-Kidney Cancer.

qABP	Target Cathepsin	Warhead	Gel Labelling	Cell Imaging	In vivo Testing	Disease	Article
BMV083	B, L, S	acyloxymethyl ketone	Y	RAW264.7	NA	NA	[156]
BMV157	S	acyloxymethyl ketone	Y	BMM	M-4T1 tumors	BC	[156]
EM053	B, L, S, X	phenoxymethyl ketone	Y	BMM	NA	NA	[156]
GB137	B, S	acyloxymethyl ketone	NA	Mac	M	AS	[153]
JF1	B	-	NA	MCF7	M	BC	[157]
Probe 4	B	Cbz-Lys-Lys(2,4-DNP)-PABA	Y	DU145, MDA-MB-231	NA	PrC, BC	[158]
AS1411-Ce6-CQDs	B	GRRGKGGC cleavage	NA	HEK293, HeLa	NA	CerC, KidC	[159]

Table 3. Photosensitization quenched Activity-Based Probes (PS-qABPs) carrying specific cleavage sequences for Cathepsin B (CtsB) are highlighted. PS-qABPs were tested for specific CtsB detection in cells in vitro, and in vivo using whole animal systems. IC₅₀ concentrations are highlighted as are their abilities to inhibit tumor proliferation. The diseases that the qABPs have been tested for are also highlighted. NA-Not-Applicable; M-Mouse; H-Human; Lun-Lung; Liv-Liver; PTX-Paclitaxel; TNBC-Triple-Negative Breast Cancer; BladC-Bladder Cancer; Xeno-Xenografts.

PS-qABP Substrates	Cathepsin-Specific Sequence	In vitro	In vivo	Cancer	IC50	Tumor Inhibition	Article
PMB1/2	B (GFLG)	A549,HepG2	NA	Lun, Liv	0.21–0.39 μM	NA	[160]
CTSB-PPP	B (GAGRRAAG)	Bone Marrow Cells	NA	NA	12 µM (71.7 ± 7.9% reduced viability)	NA	[20]
Pep-SQ@USPIO	B (GFLG)	MDA-MB-231, MCF7	M	TNBC	100–800 µg/ml	83.5 ± 8.7% (Tumor Inhibition)	[161]
NPs@Ce6	B (GFLG)	T24, Spheroids	H-M Xeno.	BladC	0.16 μg/mL (PTX/Ce-6)	5x Reduction Tumor Volume	[162]

Table 4. Selected Prodrugs are highlighted along with specific cleavage sequences for Cathepsin B (CtsB). PS-qABPs drugs were tested for their inhibitory effects in vitro cellular systems and in whole-animal systems. DOX-Doxorubicin; MIS-Minimal Inhibitory Sequence; M-Mouse; NA-Not-Applicable; BC-Breast Cancer; FibS-Fibrosarcoma; TNBC-Triple-Negative Breast Cancer; ColC-Colon cancer; Card-Cardiomyocytes; PanC-Pancreatic Cancer; PrC-Prostate Cancer; LuC-Lung Cancer; Pan-Pancreatic Epithelial; Ova-Ovarian Cancer; CerC-Cervical Cancer; GasC-Gastrointestinal Cancer; Endo-Endothelial; LivC-Liver Cancer; NSCLC-Non-Small Cell Lung Cancer.

Prodrug	Cathepsin-Specific Sequence	Drug/Nanocarrier	In vitro	In vivo	Cancer	Inhibitory Effect	Article
FRRG-DOX	B (FRRG)	DOX/ Self-assembling	HT29	M	ColC	62.4–83.1% reduction in tumor volume (1201 ± 297 to 315 ± 50 mm^3)	[170]
PNPs	B (FRRG)	DOX/Self-assembling	SKOV-3, HeyA8, MC38, CT26, H9C2, HDF, CCD-18Co	M	OvaC	IC50 9.11, 5.06, 8.98, 5.2, 111.36, 111.72, and 135.8 μM for SKOV-3, HeyA8, MC38, CT26, H9C2, HDF, and CCD-18Co cells	[167]
DPs-TPS4	B (FFKF)	DOX/ Self-assembling nanofibers	4T1, MDA-MB 231	NA	BC	NA	[172]
cPCPs	B (GAGRRAAG)	DOX/PEG	HT1080	NA	FibS	IC50 0.86 μM	[168]
GLFG/DOX	B (GLFG)	DOX/PEG Liposomes	HepG2	Z	LivC	GLFG/DOX inhibited cell viability by 60%	[173]
pHLIP-SS-GFLG-MSPLNPs@DOX	B (GFLG)	DOX/Mesoporous silica	A549, HepG2	M	LuC, LivC	90% (100 μg/ml)	[169]
Al-ProD	B (FRRG)	DOX/Albumin nano-vectors	MDA-MB231 H9C2	M	BC	7.33 µM and >200 µM for MDA-MB231 and H9C2 cells	[174]
AuNR Cluster	B (-)	DOX/Gold nano-rods	MCF-7, 16HBE	NA	LuC, BC	50%-80% (2.3 μg/ml)	[171]
MORAb-202	B (V-Cit)	Eribulin/ Maleimido-PEG2	Ovarian, Gastric and TNBC	M	OvaC, GasC, Endo, TNBC, NSCLC	IC50 20 pmol/L (for IGROV1)	[184]
BIMA,cath,KPA2	B (V-Cit)	BIM BH3 peptide/ PEG micelles	MEF, HeLa	M	CerC	~3-25 µM (50–0% viability)	[175]
PDMS-PMOXA	B (GFLG)	PAC/ Diblock-copolymer	OVCAR3	NA	OvaC	IC50 29.93 nM	[176]
Tf-MSN@AB	B (DEGFLGED)	Elevated Fe/ ROS/ Mesoporous silica	MCF7	M	BC	12–36% increased mortality	[177]
FA-GFLG-SN38	B (GFLG)	Folic acid/NA	SK-Hep-1, HeLa, Siha	NA	LuC	IC50 2–3 μM	[178]
r9-CatB-p14 MIS	B (GFLG)	P14 MIS/ NA	PK‐8, BxPC, HeLa, PC‐9, MCF‐7, T47D	M	PanC, BC, ColC, PrC, LuC	60–65% at 10‐μM	[179]
SMAC-FRRG-DOX	B (FRRG)	DOX-SMAC peptide/Self-assembling	MCF7	M	BC	Decreases in tumor volume to 71.5 ± 2.8%, 69.7 ± 3.33% and 58.6 ± 2.15% (14 d post-treatment)	[180]
PNPs	B (FRRG)	DOX (+Navitoclax)/Self- assembling	MDA-MB231	M	BC	PNPs+Navitoclax (76.97 ± 7.31% apoptotic) versus PNPs alone (47.76 ± 6.78%)	[183]
PD-NPs	B (FRRG)	DOX-anti-PD-L1 peptide/Self-assembling	HUVEC, H9C2	M	Card, BC	IC50 3.01 μM, 200 μM and >200 μM for 4T1, HUVEC and H9C2 cells	[187]
CAP-NPs	B (FRRG)	DOX (+anti-PD-L1)/Self-assembling	CT26, H9C2	M	ColC	50% complete regression compared	[186]
MTX–G–P4LDN–E2	B (GFLG)	Methotrexate-anti-EGFR peptide/P4LDN	MDA-MB-231	M	TNBC	82.6% reduced tumor volume	[181]
MORAb‐202	B (V-Cit)	Eribulin, Farletuzumab/ NA	OVCAR‐3‐A1, NCI‐H2110, A431‐A3, HL‐60, SJSA‐1	CM	BC	IC50 0.1 × 10^−10M, 7.4 × 10^−10M, and 230 × 10^−10M for IGROV‐1, NCI‐H2110, and A431‐A3 cells	[185]
HADD	B (GFLG)	Hyaluronic acid-Docetaxel/Self-assembling	MDA-MB-231	M	BC	High doses-99.71% tumor inhibition	[182]

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