RESUMEN
BACKGROUND: For cancer therapy, the identification of both selective autophagy targets and small molecules that specifically regulate autophagy is greatly needed. Heat shock protein 70 (Hsp70) is a recently discovered BH3 receptor that forms a proteinâprotein interaction (PPI) with Bcl-2-interacting mediator of cell death (Bim). Herein, a specific inhibitor of the Hsp70-Bim PPI, S1g-2, and its analog S1, which is a Bcl-2-Bim disruptor, were used as chemical tools to explore the role of Hsp70-Bim PPI in regulating mitophagy. METHODS: Co-immunoprecipitation and immunofluorescence assays were used to determine protein interactions and colocalization patterns. Organelle purification and immunodetection of LC3-II/LC3-I on mitochondria, endoplasmic reticulum (ER) and Golgi were applied to identify specific types of autophagy. Cell-based and in vitro ubiquitination studies were used to study the role of the Hsp70-Bim PPI in parkin-mediated ubiquitination of outer mitochondrial membrane 20 (TOMM20). RESULTS: We found that after the establishment of their PPI, Hsp70 and Bim form a complex with parkin and TOMM20, which in turn facilitates parkin translocation to mitochondria, TOMM20 ubiquitination and mitophagic flux independent of Bax/Bak. Moreover, S1g-2 selectively inhibits stress-induced mitophagy without interfering with basal autophagy. CONCLUSIONS: The findings highlight the dual protective function of the Hsp70-Bim PPI in regulating both mitophagy and apoptosis. S1g-2 is thus a newly discovered antitumor drug candidate that drives both mitophagy and cell death via apoptosis.
Asunto(s)
Proteínas HSP70 de Choque Térmico , Mitofagia , Proteínas HSP70 de Choque Térmico/metabolismo , Autofagia/fisiología , Mitocondrias/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.
Asunto(s)
Apoptosis , Proteína 11 Similar a Bcl2/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Pliegue de Proteína , Regulación Alostérica , Proteína 11 Similar a Bcl2/genética , Proteínas HSP70 de Choque Térmico/genética , Humanos , Células K562 , Dominios Proteicos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Although chemotherapeutics or molecular targeted drugs often elicit profound initial responses, fractional killing capable of driving acquired resistance can persist. Identifying stress-induced negative feedback or an incoherent feedforward loop (IFFL), which may contribute to fractional killing, is urgently needed. EXPERIMENTAL APPROACH: Mathematical modelling was used to identify how and to what extent a recently reported Hsp70-Bim protein-protein interaction (PPI) contributes to the adaptation of the Bcl-2 network. Experimental validation was made by using a specific inhibitor of Hsp70-Bim PPI, S1g-2, as chemical tool. Bifurcation analysis and stochastic simulation were used for the theoretical study of the impact of Hsp70-Bim PPI on cell-fate heterogeneity and factional killing. KEY RESULTS: The Hsp70-Bim-AKT circuit forms an IFFL that greatly contributes to the adaptation of the Bcl-2-regulated apoptosis network, thus leading to fractional killing. This adaptive programme enhances noise-induced cell-fate heterogeneity by shifting from a saddle-node to a saddle-collision transition scenario. CONCLUSION AND IMPLICATIONS: Hsp70-Bim IFFL serves as a molecular pathway induced by DNA damaging drugs or tyrosine kinase inhibitors that enabled fractional killing, whereby acquired resistance emerges. A synergistic strategy is unveiled for overcoming fractional killing by suppressing Hsp70-Bim PPI.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Proteínas Proto-Oncogénicas , Proteína 11 Similar a Bcl2 , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Apoptosis/genética , Línea Celular TumoralRESUMEN
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.
Asunto(s)
Proteínas HSP70 de Choque Térmico , Simulación de Dinámica Molecular , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Sitios de Unión , Dominios Proteicos , Secuencia de BasesRESUMEN
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 (Kd = 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.
Asunto(s)
Compuestos de Bifenilo , Proteínas HSP70 de Choque Térmico , Leucemia Mielógena Crónica BCR-ABL Positiva , Simulación del Acoplamiento Molecular , Humanos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/química , Compuestos de Bifenilo/farmacología , Compuestos de Bifenilo/química , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Relación Estructura-Actividad , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Proteína 11 Similar a Bcl2/metabolismo , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Unión Proteica , Descubrimiento de DrogasRESUMEN
INTRODUCTION: Myeloid leukemia 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, is an attractive target for cancer therapy. In recent years, significant progress has been made with regard to Mcl-1 inhibitors, leading to the generation of highly potent Mcl-1 inhibitors that have entered clinical trials. AREAS COVERED: This review provides an overview of the patent literature between 2020 and 2022 -covering inhibitors, antibody-drug conjugate (ADC), and proteolysis targeting chimera (PROTAC) of Mcl1. EXPERT OPINION: Despite the great success of Mcl-1 inhibitor development, the on-target toxicity to heart indicated that the BH3 mimetic Mcl-1 inhibitors could have a limited therapeutic window.Drug combinations of Mcl-1 inhibitors with targeted therapies or chemotherapies may improve safety as they may reduce the dose of Mcl-1 inhibitors. Alternatively, some technologies like ADC and PROTACS could also be utilized to improve the therapeutic window. We envision a precision medicine platform like BH3 profiling or single-molecule pull-down and co-immunoprecipitation platform will enable the tailored use of Mcl-1 inhibitors utilizing the unique molecular information of individual patients.
Asunto(s)
Antineoplásicos , Leucemia Mieloide , Humanos , Apoptosis , Patentes como Asunto , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Combinación de Medicamentos , Línea Celular Tumoral , Antineoplásicos/farmacologíaRESUMEN
Selectively targeting the cancer-specific protein-protein interaction (PPI) between Hsp70 and Bim has been discovered as a promising strategy for treating chronic myeloid leukemia (CML). The first Hsp70-Bim PPI inhibitor, S1g-2, has been identified to overcome the on-target toxicity of known Hsp70 inhibitors when it induces apoptosis of CML cells. Herein, we carried out a hit-to-lead optimization of S1g-2, yielding S1g-10, which exhibited a 10-fold increase in Hsp70/Bim suppressing potency. Furthermore, S1g-10 not only exhibited a 5- to 10-fold stronger antitumor activity in the sub-µM range against CML cells than S1g-2 in vitro, but it also overcame BCR-ABL-independent tyrosine kinase inhibitor resistance in CML in vivo depending on the Hsp70-Bim signaling pathway. Moreover, through structure-activity relationship analysis, TROSY-HSQC NMR, molecular dynamics simulation, and point mutation validation, two hydrophobic pockets composed of eight key residues were demonstrated to produce predominant interactions with either Bim or S1g-10, regarded as the "hot-spots" in the Hsp70-Bim PPI interface.
Asunto(s)
Proteínas de Fusión bcr-abl , Transducción de Señal , Apoptosis , Proteína 11 Similar a Bcl2/metabolismo , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/metabolismo , Mesilato de Imatinib/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismoRESUMEN
Targeting cancer-related Hsp70-Bim protein-protein interactions (PPIs) offers a new strategy for the design of Hsp70 inhibitors. Herein, we discovered a novel Hsp70 inhibitor, S1g-6, based on the established BH3 mimetics. S1g-6 exhibited sub-µM binding affinity toward Hsp70 and selectively disrupted Hsp70-Bim PPI. The target specificity of S1g-6in situ was validated by affinity-based protein profiling, co-immunoprecipitation, and cell-based shRNA assays. S1g-6 specifically antagonized the ATPase activity of Hsp70 upon recruiting Bim and showed selective apoptosis induction in some cancer cell lines over normal ones through suppression of some oncogenic clients of Hsp70, representing a new class of antitumor candidates. Hsp70-Bim PPI exhibited cancer-dependent role as a potential anti-cancer target.
Asunto(s)
Antineoplásicos/farmacología , Proteína 11 Similar a Bcl2/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Antineoplásicos/química , Proteína 11 Similar a Bcl2/química , Relación Dosis-Respuesta a Droga , Proteínas HSP70 de Choque Térmico/química , Humanos , Estructura Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
Herein, we screened a novel inhibitor of the Hsp70-Bim protein-protein interaction (PPI), S1g-2, from a Bcl-2 inhibitor library; this compound specifically disrupted the Hsp70-Bim PPI by direct binding to an unknown site adjacent to that of an allosteric Hsp70 inhibitor MKT-077, showing binding affinity in sub-µM concentration range. S1g-2 exhibited overall 5-10-fold higher apoptosis-inducing activity in CML cells, primary CML blasts, and BCR-ABL-transformed BaF3 cells than other cancer cells, normal lymphocytes, and BaF3 cells, illustrating Hsp70-Bim PPI driven by BCR-ABL protects CML through oncoclient proteins that enriched in three pathways: eIF2 signaling, the regulation of eIF4E and p70S6K signaling, and the mTOR signaling pathways. Moreover, S1g-2 progressively enhanced lethality along with the increase in BCR-ABL-independent TKI resistance in the K562 cell lines and is more effective in primary samples from BCR-ABL-independent TKI-resistant patients than those from TKI-sensitive patients. By comparing the underlying mechanisms of S1g-2, MKT-077, and an ATP-competitive Hsp70 inhibitor VER-155008, the Hsp70-Bim PPI was identified to be a CML-specific target to protect from TKIs through the above three oncogenic signaling pathways. The in vivo activity against CML and low toxicity endows S1g-2 a first-in-class promising drug candidate for both TKI-sensitive and resistant CML.
Asunto(s)
Proteína 11 Similar a Bcl2/antagonistas & inhibidores , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Dominios y Motivos de Interacción de Proteínas , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Proteína 11 Similar a Bcl2/genética , Proteína 11 Similar a Bcl2/metabolismo , Proliferación Celular , Proteínas de Fusión bcr-abl/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Ratones , Ratones Desnudos , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein phosphorylation is the most significant post-translational modification for regulating cellular activities, but site-specific modulation of phosphorylation is still challenging. Using three-dimensional NMR spectra, molecular dynamics simulations, and alanine mutations, we identified that the interaction network between pT69/pS70 and R106/R109 residues prevents the phosphorylation sites from exposure to phosphatase and subsequent dephosphorylation. A Bcl-2-dephosphorylation probe, S1-6e, was designed by installing a carboxylic acid group to a Bcl-2 inhibitor. The carboxyl group competitively disrupts the interaction network between R106/R109 and pT69/pS70 and subsequently facilitates Bcl-2 dephosphorylation in living cells. As a result, S1-6e manifests a more effective apoptosis induction in pBcl-2-dependent cancer cells than other inhibitors exhibiting a similar binding affinity for Bcl-2. We believe that targeting the allosteric pathways interconnecting the core-functional domain and the phosphorylation site can be a general strategy for a rational design of site-specific dephosphorylating probes, since the allosteric pathway has been discovered in a variety of proteins.