Zhichao Zhang

Developing targeted protein degradation (TPD) strategies with disease-specific mechanisms, modularity, and facile designability could ensure drug efficacy and selectivity. Herein, a small-molecule, Hsp70-based targeted protein degradation platform, termed Hsp70TAC, is described that enables tumor-selective degradation of both intracellular and extracellular proteins through distinct cellular pathways. By conjugating protein-of-interest (POI) ligands to Hsp70 inhibitors, Hsp70TACs exploits the chaperone functions of Hsp70 to enable protein degradation through both the ubiquitin-proteasome system and the endocytosis-lysosome pathway. As a proof of concept, Hsp70TACs induced efficient degradation of intracellular Bromodomain Protein 4 (BRD4) via the ubiquitin-proteasome system (DC₅₀ = 0.67 μM) and membrane-bound Programmed Death Ligand 1 (PD-L1) via caveolin-mediated endocytosis-lysosomal processing (DC₅₀ = 0.84 μM). Moreover, Hsp70TACs exploits the elevated expression of Hsp70 in tumor cells to preferentially accumulate in these cells, thereby enabling the tumor-selective degradation of POIs in Hsp70-enriched tumor cells.

The 70 kDa heat shock protein (HSP70) isoforms play distinct roles in cancer, but their structural similarity limits isoform-specific inhibition. Here, we modified nonselective HSP70 inhibitor S1g-10 via a subcellular anchoring strategy to generate compounds 5 and 8, which selectively target mitochondrial-localized GRP75 and ER-localized GRP78, respectively. Both compounds modulated their intended targets in vivo. Additionally, GRP75, but not GRP78, was identified as a key regulator of mitochondrial membrane stability in breast cancer cells and maintains the stemness of breast cancer stem cells (BCSCs). Compared with normal cells, compound 5 exhibited selective toxicity against breast cancer cells and effectively suppressed the properties of BCSCs. This study provides novel chemical probes for studying specific isoforms of HSP70 and introduces a strategy to develop GRP75-targeted therapeutics for breast cancer.

Herein, we developed a dual-factor recognition activatable probe, S1-F, which accurately lights up Bcl-2 and GSH dual-overexpressed cancer tissue with a superior tumor-to-normal tissue (T/N) ratio, highlighting the potential for this probe to be used in the visual diagnosis and imaging-guided surgery of cancer.

Hsp70-Bim protein-protein interaction (PPI) is the most recently identified specific target in chronic myeloid leukemia (CML) therapy. Herein, we developed a new class of Hsp70-Bim PPI inhibitors via scaffold hopping of S1g-10, the most potent Hsp70-Bim PPI inhibitor thus far. Through structure-activity relationship (SAR) study, we obtained a biphenyl scaffold compound JL-15 with a 5.6-fold improvement in Hsp70-Bim PPI suppression (K_d = 123 vs 688 nM) and a 4-fold improvement in water solubility (29.42 vs 7.19 μg/mL) compared to S1g-10. It maintains comparable apoptosis induction capability with S1g-10 against both TKI-sensitive and TKI-resistant CML cell lines in an Hsp70-Bim-dependent manner. Additionally, through SAR, ¹H-¹⁵N TRSOY-NMR, and molecular docking, we revealed that Lys319 is a "hot spot" in the Hsp70-Bim PPI interface. Collectively, these results provide a novel chemical scaffold and structural insights for the rational design of Hsp70-Bim PPI inhibitors.

The process of degrading unwanted or damaged mitochondria by autophagy, called mitophagy, is essential for mitochondrial quality control together with mitochondrial apoptosis. In mammalian cells, pan-Bcl-2 family members including conical Bcl-2 members and non-conical ones are involved in and govern the two processes. We have illustrated recently the BH3 receptor Hsp70 interacts with Bim to mediate both apoptosis and mitophagy. However, whether similar pathways exist in lower eukaryotes where conical Bcl-2 members are absent remained unclear. Here, a specific inhibitor of the Hsp70-Bim PPI, S1g-10 and its analogs were used as chemical tools to explore the role of yeast Bxi1/Ybh3 in regulating mitophagy and apoptosis. Using Om45-GFP processing assay, we illustrated that yeast Ybh3 mediates a ubiquitin-related mitophagy pathway in both yeast and mammalian cells through association with Hsp70, which is in the same manner with Bim. Moreover, by using Bax/Bak double knockout MEF cells, Ybh3 was identified to induce apoptosis through forming oligomerization to trigger mitochondrial outer membrane permeabilization (MOMP) like Bax. We not only illustrated a conserved ubiquitin-related mitophagy pathway in yeast but also revealed the multi-function of Ybh3 which combines the function of BH3-only protein and multi-domain Bax protein as one.

Introduction: Estrogen receptor (ER) positive patients compromise about 70% of breast cancers. Tamoxifen, an antagonist of ERα66 (the classic ER), is the most effective and the standard first-line drug. However, its efficacy is limited by the development of acquired resistance. Methods: A specific inhibitor of Hsp70-Bim protein–protein interaction (PPI), S1g-2, together with an inhibitor of Hsp70-Bag3 PPI, MKT-077 and an ATP-competitive inhibitor VER155008, were used as chemical tools. Cell viability assays, co-immunoprecipitation and gene knockdown were used to investigate the role of Hsp70 in tamoxifen resistance. A xenograft model was established in which tamoxifen-resistant breast cancer (MCF-7/TAM-R) cells maintained in the presence of 5 μM tamoxifen were subcutaneously inoculated. The anti-tumor efficiency of S1g-2 was measured after a daily injection of 0.8 mg/kg for 14 days. Results: It was revealed that Hsp70-Bim PPI protects ERα-positive breast cancer from tamoxifen-induced apoptosis through binding and stabilizing ERα36, rather than ERα66, resulting in sustained EGFR mRNA and protein expression. Disruption of Hsp70-Bim PPI and downregulation of ERα36 expression in tumor samples are consistent with the in vitro functions of S1g-2, resulting in about a three-fold reduction in tumor volume. Conclusions: The in vivo activity and safety of S1g-2 illustrated that it is a potential strategy for Hsp70-Bim disruption to overcome tamoxifen-resistant ER-positive breast cancer.

Context The interface of Hsp70-Bim protein–protein interaction (PPI) has been identified as a specific target for Chronic Myeloid Leukemia (CML) therapy and the specific inhibitors were developed to exhibit in vivo anti-leukemia activities. Herein, we explored the binding mechanism of a Hsp70-Bim inhibitor, 6-(cyclohexylthio)-3-((2-morpholinoethyl) amino)-1-oxo-1H-phenalene-2-carbonitrile (S1g-6), to Hsp70 at the atomic level by MD simulation. TYR-149, THR-222, ALA-223, and GLY-224 on Hsp70 were identified as four key residues that contribute to Hsp70/S1g-6 complex. Moreover, the site mutation validation demonstrated the TYR-149 of Hsp70 is a “hot-spot” in the Hsp70-Bim PPI interface. These results could benefit the design of further inhibitors to occupy the Bim binding site on the Hsp70 surface. Methods The binding mechanism of S1g-6 and Hsp70 was predicted through the molecular dynamics (MD) method by Gromacs-2021.3. The MD simulation was performed with 100-ps NVT and 100-ps NPT ensemble, and the force field was chosen as the Charmm36 force field. The temperature was set as 300 K, the time step was 2 fs and the total MD simulation time was 500 ns.