Probing Allosteric Hsp70 Inhibitors by Molecular Modelling Studies to Expedite the Development of Novel Combined F508del CFTR Modulators

Cystic fibrosis (CF) is caused by different mutations related to the cystic fibrosis transmembrane regulator protein (CFTR), with F508del being the most common. Pioneering the development of CFTR modulators, thanks to the development of effective correctors or potentiators, more recent studies deeply encouraged the administration of triple combination therapeutics. However, combinations of molecules interacting with other proteins involved in functionality of the CFTR channel recently arose as a promising approach to address a large rescue of F508del-CFTR. In this context, the design of compounds properly targeting the molecular chaperone Hsp70, such as the allosteric inhibitor MKT-077, proved to be effective for the development of indirect CFTR modulators, endowed with ability to amplify the accumulation of the rescued protein. Herein we performed structure-based studies of a number of allosteric HSP70 inhibitors, considering the recent X-ray crystallographic structure of the human enzyme. This allowed us to point out the main interaction supporting the binding mode of MKT-077, as well as of the related analogues. In particular, cation-π and π–π stacking with the conserve residue Tyr175 deeply stabilized inhibitor binding at the HSP70 cavity. Molecular docking studies had been followed by QSAR analysis and then by virtual screening of aminoaryl thiazoles (I–IIIa) as putative HSP70 inhibitors. Their effectiveness as CFTR modulators has been verified by biological assays, in combination with VX-809, whose positive results confirmed the reliability of the whole applied computational method. Along with this, the “in-silico” prediction of absorption, distribution, metabolism, and excretion (ADME) properties highlighted, once more, that AATs may represent a chemical class to be further investigated for the rational design of novel combination of compounds for CF treatment.

Similarities and Differences of Hsp70, hsc70, Grp78 and Mortalin as Cancer Biomarkers and Drug Targets

Background: Upregulation of Heath Shock Protein 70 (HSP70) chaperones supports cancer cell survival. Their high homology causes a challenge to differentiate them in experimental or prevention and treatment strategies. The objective of this investigation was to determine similarities and differences of Hsp70, hsc70, Grp78 and Mortalin members of the HSP70 family encoded by HSPA1, HSPA8, HSPA5 and HSPA9 genes, respectively. Methods: Literature reviews were conducted using HSPA1, HSPA5, HSPA8 and HSPA9 gene or protein names or synonyms combined with biological or cancer-relevant terms. Ingenuity Pathway Analysis was used to identify and compare profiles of proteins that directly bind individual chaperones and their associated pathways. TCGA data was probed to identify associations of hsc70 with cancer patient survival. ClinicalTrials.gov was used to identify HSP70 family studies. Results: The chaperones have similar protein folding functions. Their different cellular effects are determined by co-chaperones and client proteins combined with their intra- and extra-cellular localizations. Their upregulation is associated with worse patient prognosis in multiple cancers and can stimulate tumor immune responses or drug resistance. Their inhibition selectively kills cancer over healthy cells. Conclusions: Differences in Hsp70, hsc70, Grp78 and mortalin provide opportunities to calibrate HSP70 inhibitors for individual cancers and combination therapies.

HSP70 Inhibition Blocks Adaptive Resistance and Synergizes with MEK Inhibition for the Treatment of NRAS-Mutant Melanoma

NRAS-mutant melanoma is currently a challenge to treat. This is due to an absence of inhibitors directed against mutant NRAS, along with adaptive and acquired resistance of this tumor type to inhibitors in the MAPK pathway. Inhibitors to MEK have shown some promise for NRAS-mutant melanoma. In this work, we explored the use of MEK inhibitors for NRAS-mutant melanoma. At the same time, we investigated the impact of the brain microenvironment, specifically astrocytes, on the response of a melanoma brain metastatic cell line to MEK inhibition. These parallel avenues led to the surprising finding that astrocytes enhance the sensitivity of melanoma tumors to MEK inhibitors (MEKi). We show that MEKi cause an upregulation of the transcriptional regulator ID3, which confers resistance. This upregulation of ID3 is blocked by conditioned media from astrocytes. We show that silencing ID3 enhances the sensitivity of melanoma to MEKi, thus mimicking the effect of the brain microenvironment. Moreover, we report that ID3 is a client protein of the chaperone HSP70, and that HSP70 inhibition causes ID3 to misfold and accumulate in a detergent-insoluble fraction in cells. We show that HSP70 inhibitors synergize with MEKi against NRAS-mutant melanoma, and that this combination significantly enhances the survival of mice in two different models of NRAS-mutant melanoma. These studies highlight ID3 as a mediator of adaptive resistance, and support the combined use of MEK and HSP70 inhibitors for the therapy of NRAS-mutant melanoma. Significance: MEKi are currently used for NRAS-mutant melanoma, but have shown modest efficacy as single agents. This research shows a synergistic effect of combining HSP70 inhibitors with MEKi for the treatment of NRAS mutant melanoma.

Еvaluation of antitumor activity of some 4-aminopiperidine derivatives — low molecular weight Hsp70 inhibitors — on transplantable mouse tumors

Low molecular weight compounds targeting chaperone proteins Hsp90 and Hsp70 have opened up a new avenue in the therapy of neoplasms. In 2020, we tested 3 Hsp70 inhibitors from the class of 4-aminopiperidine derivatives for their antitumor activity on in vivo models. The list of the tested compounds included N-(2-chlorobenzyl)-N-ethyl-1-(2-(methylthio)pyrimidin-4-yl)piperidin-4-amine (compound 1), 4-((methyl(1-(2-(methylthio)pyrimidin-4-yl) piperidin-4-yl)amino)methyl) benzonitrile (compound 2) and N-(2,6- dichlorobenzyl)-1-(1-(2-(ethylthio)pyrimidin-4-yl)piperidin-4-yl)-N-methylmethaneamine (compound 3). The aim of this study was to compare the efficacy of 4-aminopiperidine derivatives in vivo using the models of transplantable murine L1210 lymphocytic leukemia and B16 melanoma. Compounds 2 and 3 used in combination with cyclophosphamide exhibited high cytotoxic activity (р = 0.05) against L1210 leukemia (an 80-82% increase in survival time) and B16 melanoma (98-99.7% tumor growth delay). For L1210 lymphocytic leukemia, compounds 2 and 3 used in combination with cyclophosphamide fell into the low (+) therapeutic potential category. For B16 melanoma, compounds 1, 2 and 3 used in combination with cyclophosphamide fell into either low (+) or moderate (++) therapeutic potential categories. On the whole, the tested doses of the compounds used in combination with cyclophosphamide hold promise for the therapy of L1210 leukemia and B16 melanoma in mouse models. Our findings confirm the potential of low molecular weight Hsp70 inhibitors for combination chemotherapy against cancer.

Development of Novel Rhodacyanine-Based Heat Shock Protein 70 Inhibitors

Background: A growing body of evidence suggests that Hsp70, which is overexpressed in human breast tumors, plays a role in tumorigenesis and tumor progression in breast cancer as well as in its aggressive phenotypes. Hsp70 constitutes a potential therapeutic target in the treatment of this disease. Method: We developed a new series of rhodacyanine-based Hsp70 inhibitors, represented by compounds 1 and 6, in which the cationic pyridin-1-ium or thiazol-3-ium ring of existing Hsp70 inhibitors (e.g., JG-40 and JG-98) was replaced by a corresponding benzo-fused N-heterocycle. Results: Several lines of evidence suggest that these benzo-fused derivatives may exert their antitumor activities, in part, by targeting Hsp70. These putative inhibitors displayed differential antiproliferative efficacy against breast cancer cells (IC50 as low as 0.25 µM) versus nontumorigenic MCF-10A breast epithelial cells (IC50 ≥ 5 µM). This was correlated with the corresponding Hsp70 expression levels. Using a protein refolding assay, we confirmed that these agents effectively inhibited the chaperone activity of Hsp70. Moreover, these inhibitors effectively suppressed the expression of well-known oncogenic client proteins of Hsp70’s, including FoxM1, HuR, and Akt, which paralleled their antiproliferative efficacy. Supporting the established role of Hsp70 in regulating protein refolding, these derivatives induced autophagy, as manifested by the conversion of LC3B-I to LC3B-II. Notably, these putative Hsp70 inhibitors did not cause a compensatory elevation in Hsp90 expression, contrasting with the previously reported effects of Hsp90 inhibitors on Hsp70 upregulation. Conclusion: Together with the finding that compounds 1 and 6 showed improved microsomal stability, these results suggest the translational potential of these putative Hsp70 inhibitors to foster new strategies for cancer therapy. However, whether these benzo-fused rhodacyanines act on kinases or other targets remains unclear, which is currently under investigation.

Allosteric HSP70 inhibitors perturb mitochondrial proteostasis and overcome proteasome inhibitor resistance in multiple myeloma

Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first map proteasome-associated genetic co-dependencies. We identify cytosolic heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma tumor cells overcoming PI-induced stress. These results lead us to explore allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. We show these compounds exhibit increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Surprisingly, shotgun and pulsed-SILAC proteomics reveal that JGs overcome PI resistance not via the expected mechanism of inhibiting cytosolic HSP70s, but instead through mitochondrial-localized HSP70, HSPA9, destabilizing the 55S mitoribosome. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize dynamics of myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.

Therapeutic potency of heat-shock protein-70 in the pathogenesis of colorectal cancer: current status and perspectives

Heat-shock protein-70 (HSP70) is critical to the folding, stability, and activity of several client proteins including many responsible for cancer cell proliferation, apoptosis, drug toxicity, and metastasis. Up-regulation of HSP70 is positively associated with increased tumorigenicity as well as poor survival in colon cancer patients, supporting the diagnostic, prognostic, and therapeutic potencies of HSP70 in colorectal cancer. The administration of specific pharmacological inhibitors or gene knock-down for HSP70 suppresses tumor progression and enhances tumor cell chemosensitivity. This review summarizes the different tumorigenic properties of HSP70 and the potential therapeutic potency of HSP70 inhibitors in terms of a novel strategy for colorectal cancer therapy, for a better understanding, and hence better management of this disease.

HSP70 Inhibitor Suppresses IGF-I-Stimulated Migration of Osteoblasts through p44/p42 MAP Kinase

Heat shock protein 70 (HSP70) is a ubiquitously expressed molecular chaperone in a variety of cells including osteoblasts. We previously showed that insulin-like growth factor‐I (IGF-I) elicits migration of osteoblast-like MC3T3-E1 cells through the activation of phosphatidylinositol 3-kinase/Akt and p44/p42 mitogen-activated protein (MAP) kinase. In the present study, we investigated the effects of HSP70 inhibitors on the IGF-I-elicited migration of these cells and the mechanism involved. The IGF-I-stimulated osteoblast migration evaluated by a wound-healing assay and by a transwell cell migration was significantly reduced by VER-155008 and YM-08, which are both HSP70 inhibitors. VER-155008 markedly suppressed the IGF-I-induced phosphorylation of p44/p42 MAP kinase without affecting that of Akt. In conclusion, our results strongly suggest that the HSP70 inhibitor reduces the IGF-I-elicited migration of osteoblasts via the p44/p42 MAP kinase.