3D-QSAR and Molecular Docking Approaches for the Identification of Phyto-Inhibitors of Hsp90

Inhibition of Hsp90 disrupts the Hsp90 client protein complex, resulting in its breakdown. Phytochemicals from reported anticancer plants were screened against the orthosteric site of Hsp90. The lead compounds were subjected to the Lipinski rule of five to evaluate their drug-likeness. Three-Dimensional Quantitative Structure-Activity Relationships (3D-QSAR), a mathematical model for the inhibition of Hsp90, was also derived. The lead compounds are guaiol from Cannabis sativa, actinidine from Anacadium occidentale, and choline from Tinospora cordifolia with docking scores of -11kcal/mol, -12.1kcal/mol, and -10.8kcal/mol, respectively. The 3D-QSAR model generated is robust and thoroughly validated with a correlation coefficient R of 0.94 and R2 of 0.950. Actinidine, choline and, guaiol are novel and potent inhibitors of Hsp90. They form interactions with key amino acid residues within the Hsp90 orthosteric site.

HSP-90/kinase complexes are stabilized by the large PPIase FKB-6

Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery.

Novel Small Molecule Hsp90/Cdc37 Interface Inhibitors Indirectly Target K-Ras-Signaling

The ATP-competitive inhibitors of Hsp90 have been tested predominantly in kinase addicted cancers; however, they have had limited success. A mechanistic connection between Hsp90 and oncogenic K-Ras is not known. Here, we show that K-Ras selectivity is enabled by the loss of the K-Ras membrane nanocluster modulator galectin-3 downstream of the Hsp90 client HIF-1α. This mechanism suggests a higher drug sensitivity in the context of KRAS mutant, HIF-1α-high and/or Gal3-high cancer cells, such as those found, in particular, in pancreatic adenocarcinoma. The low toxicity of conglobatin further indicates a beneficial on-target toxicity profile for Hsp90/Cdc37 interface inhibitors. We therefore computationally screened >7 M compounds, and identified four novel small molecules with activities of 4 μM–44 μM in vitro. All of the compounds were K-Ras selective, and potently decreased the Hsp90 client protein levels without inducing the heat shock response. Moreover, they all inhibited the 2D proliferation of breast, pancreatic, and lung cancer cell lines. The most active compounds from each scaffold, furthermore, significantly blocked 3D spheroids and the growth of K-Ras-dependent microtumors. We foresee new opportunities for improved Hsp90/Cdc37 interface inhibitors in cancer and other aging-associated diseases.

Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability

The E3 ubiquitin-ligase Hakai binds to several tyrosine-phosphorylated Src substrates, including the hallmark of the epithelial-to-mesenchymal transition E-cadherin, and signals for degradation of its specific targets. Hakai is highly expressed in several human cancers, including colon cancer, and is considered as a drug target for cancer therapy. Here, we report a link between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the correct folding of its client proteins, allowing them to maintain their stability and activity. Hsp90 inhibitors specifically interfere with the association with its Hsp90 client proteins, and exhibit potent anti-cancer properties. By immunoprecipitation, we present evidence that Hakai interacts with Hsp90 chaperone complex in several epithelial cells and demonstrate that is a novel Hsp90 client protein. Interestingly, by overexpressing and knocking-down experiments with Hakai, we identified Annexin A2 as a Hakai-regulated protein. Pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Interestingly, geldanamycin-induced Hakai degradation is accompanied by an increased expression of E-cadherin and Annexin A2. We also show that geldanamycin suppresses cell motility at least in part through its action on Hakai expression. Taken together, our results identify Hakai as a novel Hsp90 client protein and shed light on the regulation of Hakai stability. Our results open the possibility to the potential use of Hsp90 inhibitors for colorectal cancer therapy through its action on Hakai client protein of Hsp90.

Targeting the MYC Oncogene in Burkitt Lymphoma through HSP90 Inhibition

Overexpression of the MYC oncogene is a key feature of many human malignancies including Burkitt lymphoma. While MYC is widely regarded to be a promising therapeutic target, a clinically effective MYC inhibitor is still elusive. Here, we report an alternative strategy, targeting MYC indirectly through inhibition of the HSP90 machinery. We found that inhibition of HSP90 function reduces MYC expression in human Burkitt lymphoma through suppression of MYC transcription and destabilization of MYC protein, thereby diminishing the proliferation of tumor cells. Consistently, treatment of Burkitt lymphoma cell lines with HSP90 inhibitors (17-AAG or 17-DMAG) was accompanied by downregulation of canonical MYC target genes. Combination treatment with 17-DMAG and the proteasome inhibitor, MG-132, led to accumulation of MYC protein, indicating that upon HSP90 inhibition, MYC is degraded by the proteasome. Using co-immunoprecipitation, we furthermore demonstrated a direct interaction between MYC and HSP90, indicating that MYC is an HSP90 client protein in Burkitt lymphoma. Together, we report here the use of HSP90 inhibitors as an alternative approach to target the MYC oncogene and its network in Burkitt lymphoma.

HSP90 promotes Burkitt lymphoma cell survival by maintaining tonic B-cell receptor signaling

Key Points HSP90 inhibition induces apoptosis in BL cells by disrupting tonic BCR signaling. SYK is an HSP90 client protein, and BCR signaling-dependent phosphorylation of HSP90 on Y197 is required for this interaction.

NPM1-TYK2 Fusion Is an Oncogene and a Novel HSP90-Client Protein

Cutaneous CD30-positive lymphoproliferative disorders (LPDs), the second most common type of cutaneous T-cell lymphoma include a clinicopathologic spectrum of benign lymphomatoid papulosis (LYP) and primary cutaneous anaplastic large-cell lymphoma (ALCL). NPM1-TYK2 is the first identified tyrosine kinase chromosomal translocation in CD30-positive LPDs. Understanding the deregulated functions resulting from new chromosomal translocations in these neoplasms is essential to discover novel therapeutic approaches. Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein, which functions as a molecular chaperone for proteins and nucleic acids. Tyrosine kinase 2 (TYK2) is a non-receptor tyrosine kinase that belongs to the family of Janus Kinases (JAKs). NPM1-TYK2 is an 81 kDa fusion protein comprising of NPM1 (1-257 amino acids) and the kinase domain of TYK2 (726-1187 amino acids). Since NPM1-TYK2 is a recently discovered fusion gene, we conducted experiments to determine its oncogenic potential. Transduction of lentiviral particles bearing NPM1-TYK2 gene transformed the interleukin-3 (IL-3) dependent Ba/F3 cell line to IL-3 independent growth through constitutive activation of TYK2 kinase and downstream STAT signaling. We further evaluated the role of NPM1 as part of fusion protein in NPM1-TYK2 mediated oncogenicity. Transfection of NPM1-TYK2 deletion construct lacking NPM1 in HEK293 cells resulted in significant decrease in TYK2 kinase activity in the cells compared to full-length NPM1-TYK2 transfected cells. Currently, there are no effective small molecule inhibitors for NPM1/ TYK2 in the clinical trials. Hence, there is a clinical unmet need to develop novel, targeted therapies for NPM1-TYK2 driven lymphomas. As TYK2 is an established HSP90 client protein, the rationale for this study is to investigate the ability of HSP90 to regulate the stability and kinase activity of NPM1-TYK2 fusion protein. HSP90 is a master chaperone involved in the proper folding and maturation of a variety of oncogenic kinases and perturbing HSP90 function is emerging as a promising approach for cancer therapy. We used Myla cells (expressing endogenous NPM1-TYK2 fusion gene) to investigate the status of NPM1-TYK2 as a HSP90 client protein. Here we report that the treatment of cells with small molecule HSP90 inhibitor, 17-AAG promotes degradation of NPM1-TYK2 fusion protein resulting in downregulation of its oncogenically induced downstream STAT signaling pathways. Mechanistically, dephosphorylation of STAT1, 3, and 5 following 17-AAG treatment resulted in apoptotic cell death of the lymphoma cells. The results obtained using 17-AAG, were further corroborated using other HSP90 small molecule inhibitors AUY922 and PU-H71. Immunoprecipitation studies clearly demonstrate that 17-AAG treatment disrupts interaction between HSP90 and NPM1-TYK2 chimeric protein. Collectively, these findings provide an evidence for NPM1-TYK2 oncogenicity and therapeutic potential of HSP90 inhibitors for the treatment of a subset of cutaneous CD30-positive lymphoproliferative disorder patients expressing NPM1-TYK2 chimeric gene. Disclosures Ganguly: Onyx: Speakers Bureau; Seattle Genetics: Speakers Bureau; Janssen: Research Funding.

Establishment of human OCT4 as a putative HSP90 client protein : a case for HSP90 chaperoning pluripotency

The therapeutic potential of stem cells is already being harnessed in clinical trails. Of even greater therapeutic potential has been the discovery of mechanisms to reprogram differentiated cells into a pluripotent stem cell-like state known as induced pluripotent stem cells (iPSCs). Stem cell nature is governed and maintained by a hierarchy of transcription factors, the apex of which is OCT4. Although much research has elucidated the transcriptional regulation of OCT4, OCT4 regulated gene expression profiles and OCT4 transcriptional activation mechanisms in both stem cell biology and cellular reprogramming to iPSCs, the fundamental biochemistry surrounding the OCT4 transcription factor remains largely unknown. In order to analyze the biochemical relationship between HSP90 and human OCT4 we developed an exogenous active human OCT4 expression model with human OCT4 under transcriptional control of a constitutive promoter. We identified the direct interaction between HSP90 and human OCT4 despite the fact that the proteins predominantly display differential subcellular localizations. We show that HSP90 inhibition resulted in degradation of human OCT4 via the ubiquitin proteasome degradation pathway. As human OCT4 and HSP90 did not interact in the nucleus, we suggest that HSP90 functions in the cytoplasmic stabilization of human OCT4. Our analysis suggests HSP90 inhibition inhibits the transcriptional activity of human OCT4 dimers without affecting monomeric OCT4 activity. Additionally our data suggests that the HSP90 and human OCT4 complex is modulated by phosphorylation events either promoting or abrogating the interaction between HSP90 and human OCT4. Our data suggest that human OCT4 displays the characteristics describing HSP90 client proteins, therefore we identify human OCT4 as a putative HSP90 client protein. The regulation of the transcription factor OCT4 by HSP90 provides fundamental insights into the complex biochemistry of stem cell biology. This may also be suggestive that HSP90 not only regulates stem cell biology by maintaining routine cellular homeostasis but additionally through the direct regulation of pluripotency factors.

Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis

The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of β-actin and γ-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components.