Glucose Transporter-Targeting Chimeras Enabling Tumor-Selective Degradation of Secreted and Membrane Proteins.

Tumor-selective degradation of target proteins has the potential to offer superior therapeutic benefits with maximized therapeutic windows and minimized off-target effects. However, the development of effective lysosome-targeted degradation platforms for achieving selective protein degradation in tumors remains a substantial challenge. Cancer cells depend on certain solute carrier (SLC) transporters to acquire extracellular nutrients to sustain their metabolism and growth. This current study exploits facilitative glucose transporters (GLUTs), a group of SLC transporters widely overexpressed in numerous types of cancer, to drive the endocytosis and lysosomal degradation of target proteins in tumor cells. GLUT-targeting chimeras (GTACs) were generated by conjugating multiple glucose ligands to an antibody specific for the target protein. We demonstrate that the constructed GTACs can induce the internalization and lysosomal degradation of the extracellular and membrane proteins streptavidin, tumor necrosis factor-alpha (TNF-α), and human epidermal growth factor receptor 2 (HER2). Compared with the parent antibody, the GTAC exhibited higher potency in inhibiting the growth of tumor cells in vitro and enhanced tumor-targeting capacity in a tumor-bearing mouse model. Thus, the GTAC platform represents a novel degradation strategy that harnesses an SLC transporter for tumor-selective depletion of secreted and membrane proteins of interest.

Lysosome-targeting chimeras containing an endocytic signaling motif trigger endocytosis and lysosomal degradation of cell-surface proteins.

Lysosome-targeting degradation technologies have emerged as a promising therapeutic strategy for the selective depletion of target extracellular and cell-surface proteins by harnessing a cell-surface effector protein such as lysosome-targeting receptors (LTRs) or transmembrane E3 ligases that direct lysosomal degradation. We recently developed a lysosome-targeting degradation platform termed signal-mediated lysosome-targeting chimeras (SignalTACs) that functions independently of an LTR or E3 ligase; these are engineered fusion proteins comprising a target binder, a cell-penetrating peptide (CPP), and a lysosomal sorting signal motif (P1). Herein, we present the next-generation SignalTACs containing a single endocytic signal that bypasses the need for a CPP. We demonstrate that the fusion with a 10-amino acid endocytic signaling peptide (P3) derived from the cation-independent mannose-6-phosphate receptor (CI-M6PR) induces robust internalization and lysosomal degradation of the target protein. The P3-based SignalTAC exhibited enhanced antitumor efficacy compared to the parent antibody. We envision that the fusion of the endocytic signaling peptide P3 to a target binder may allow the construction of an effective degrader for membrane-associated targets. Furthermore, mechanistic studies identified different drivers for the activities of the P3- and P1-based SignalTACs, which is expected to provide crucial insights toward the harnessing of the intrinsic signaling pathways to direct protein trafficking and degradation.

Harnessing the Lysosomal Sorting Signals of the Cation-Independent Mannose-6-Phosphate Receptor for Targeted Degradation of Membrane Proteins.

Membrane proteins are a crucial class of therapeutic targets that remain challenging to modulate using traditional occupancy-driven inhibition strategies or current proteolysis-targeting degradation approaches. Here, we report that the inherent endolysosomal sorting machinery can be harnessed for the targeted degradation of membrane proteins. A new degradation technique, termed signal-mediated lysosome-targeting chimeras (SignalTACs), was developed by genetically fusing the signaling motif from the cation-independent mannose-6-phosphate receptor (CI-M6PR) to a membrane protein binder. Antibody-based SignalTACs were constructed with the CI-M6PR signal peptides fused to the C-terminus of both heavy and light chains of IgG. We demonstrated the scope of this platform technology by degrading five pathogenesis-related membrane proteins, including HER2, EGFR, PD-L1, CD20, and CD71. Furthermore, two simplified constructs of SignalTACs, nanobody-based and peptide-based SignalTACs, were created and shown to promote the lysosomal degradation of target membrane proteins. Compared to the parent antibodies, SignalTACs exhibited significantly higher efficiency in inhibiting tumor cell growth both in vitro and in vivo. This work provides a simple, general, and robust strategy for degrading membrane proteins with molecular precision and may represent a powerful platform with broad research and therapeutic applications.

Indole-Based Long-Wavelength Fluorescent Probes for Bioimaging of S-Nitrosylation in Mitochondria.

S-Nitrosylation has been found to play an important role in regulating mitochondrial function. However, probes for detection of protein S-nitrosylation in mitochondria remain unexplored. Herein, a novel 4-(pyridin-4-yl)vinyl-substituted indole was designed, exhibiting a long-wavelength emission and a high fluorescent quantum yield. Functionalization of the 7-position of the indole ring with an arylphosphine ester resulted with probes with efficient mitochondria-targeting ability. Furthermore, the indole-arylphosphine displayed a significant fluorescence enhancement upon exposure to S-nitrosoglutathione (GSNO) at low micromolar concentrations in A431 cells. Taken together, this study provides a new indole-based fluorescent probe with a unique long-wavelength emission for direct detection of S-nitrosylation in mitochondria, which may represent a powerful tool for understanding the critical roles of S-nitrosylation within mitochondria of living organisms.

Antibody-Drug Conjugates Targeting the Human Epidermal Growth Factor Receptor Family in Cancers.

Members of the human epidermal growth factor receptor (HER) family, which includes HER1 (also known as EGFR), HER2, HER3 and HER4, have played a central role in regulating cell proliferation, survival, differentiation and migration. The overexpression of the HER family has been recognized as one of the most common cellular dysregulation associated with a wide variety of tumor types. Antibody-drug conjugates (ADCs) represent a new and promising class of anticancer therapeutics that combine the cancer specificity of antibodies with cytotoxicity of chemotherapeutic drugs. Two HER2-directed ADCs, trastuzumane-emtansine (T-DM1) and trastuzumab-deruxtecan (DS-8201a), have been approved for HER2-positive metastatic breast cancer by the U.S. Food and Drug Administration (FDA) in 2013 and 2019, respectively. A third HER2-directed ADC, disitamab vedotin (RC48), has been approved for locally advanced or metastatic gastric or gastroesophageal junction cancer by the NMPA (National Medical Products Administration) of China in 2021. A total of 11 ADCs that target HER family receptors (EGFR, HER2 or HER3) are currently under clinical trials. In this review article, we summarize the three approved ADCs (T-DM1, DS-8201a and RC48), together with the investigational EGFR-directed ADCs (ABT-414, MRG003 and M1231), HER2-directed ADCs (SYD985, ARX-788, A166, MRG002, ALT-P7, GQ1001 and SBT6050) and HER3-directed ADC (U3-1402). Lastly, we discuss the major challenges associated with the development of ADCs, and highlight the possible future directions to tackle these challenges.