Molecular Docking and Simulation Analysis of Cyclopeptides as Anticancer Agents

Background: Cancer is a leading cause of death for people worldwide, in addition to the rise in mortality rates attributed to the Covid epidemic. This allows scientists to do additional research. Here, we have selected Integerrimide A, cordy heptapeptide, and Oligotetrapeptide as the three cyclic proteins that will be further studied and investigated in this context.Methods: Docking research was carried out using the protein complexes 1FKB and 1YET, downloaded from the PDB database and used in the docking investigations. Cyclopeptides have been reported to bind molecularly to human HSP90 (Heat shock protein) and FK506. It was possible to locate HSP90 in Protein Data Banks 1YET and 1FKB. HSP90 was retrieved from Protein Data Bank 1YET and 1FKB. Based on these findings, it is possible that the anticancer effects of Int A, Cordy, and Oligo substances could be due to their ability to inhibit the mTOR rapamycin binding domain and the HSP90 Geldanamycin binding domain via the mTOR and mTOR chaperone pathways. During the calculation, there were three stages: system development, energy reduction, and molecular dynamics (also known as molecular dynamics). Each of the three compounds demonstrated a binding affinity for mTOR's Rapamycin binding site that ranged from -6.80 to -9.20 Kcal/mol (FKB12).Results: An inhibition constant Ki of 181.05 nM characterized Cordy A with the highest binding affinity (-9.20 Kcal/mol). Among the three tested compounds, Cordy A was selected for MD simulation. HCT116 and B16F10 cell lines were used to test each compound's anticancer efficacy. Doxorubicin was used as a standard drug. The cytotoxic activity of substances Int A, Cordy A, and Oligo on HCT116 cell lines was found to be 77.65 μM, 145.36 μM, and 175.54 μM when compared to Doxorubicin 48.63 μM, similarly utilizing B16F10 cell lines was found to be 68.63 μM, 127.63 μM, and 139.11 μM to Doxorubicin 45.25 μM.Conclusion: Compound Cordy A was more effective than any other cyclic peptides tested in this investigation.

Tenth International Symposium on the Hsp90 chaperone machine : Switzerland, October 19-23, 2022.

Hsp90 is a molecular chaperone responsible for regulating proteostasis under physiological and pathological conditions. Its central role in a range of diseases and potential as a drug target has focused efforts to understand its mechanisms and biological functions and to identify modulators that may form the basis for therapies. The 10th international conference on the Hsp90 chaperone machine was held in Switzerland in October 2022. The meeting was organized by Didier Picard (Geneva, Switzerland) and Johannes Buchner (Garching, Germany) with an advisory committee of Olivier Genest, Mehdi Mollapour, Ritwick Sawarkar, and Patricija van Oosten-Hawle. This was a much anticipated first in-person meeting of the Hsp90 community since 2018 after the COVID-19 pandemic led to the postponement of the 2020 meeting. The conference remained true to the tradition of sharing novel data ahead of publication, providing unparalleled depth of insight for both experts and newcomers to the field.

Role of heat shock protein 90 as an antiviral target for swine enteric coronaviruses.

A variety of swine enteric coronaviruses (SECoVs) have emerged and are prevalent in pig populations, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome (SADS)-CoV, a newly identified bat-origin CoV with zoonotic potential. Unfortunately, available traditional, inactivated and attenuated SECoV vaccines are of limited efficacy against the variants currently circulating in most pig populations. In this study, we evaluated the role of host factor heat shock protein 90 (Hsp90) as an antiviral target against SECoVs, exemplified by SADS-CoV. Pharmacological inhibition of Hsp90 diminished SADS-CoV replication significantly in porcine and human cell lines, and also decreased replication of SADS-CoV in a porcine intestinal enteroid model. Further mechanistic experiments revealed that both porcine and human isoforms of Hsp90 interact with the SADS-CoV nucleocapsid (N) protein, and inhibition of Hsp90 resulted in autophagic degradation of N protein. Moreover, we linked Hsp90 to virus-induced cellular pyroptosis, as SADS-CoV was found to trigger caspase-1/gasdermin-d-mediated pyroptotic cell death, which was mitigated by inhibition of Hsp90. Finally, we demonstrated that Hsp90 also associated with N proteins and was involved in propagation of PEDV, PDCoV and TGEV. This study thus extends our understanding of immune responses to SADS-CoV infection and offers a new potential therapeutic option against four SECoVs.

Heat shock protein 90 facilitates SARS-CoV-2 structural protein-mediated virion assembly and promotes virus-induced pyroptosis.

Inhibition of heat shock protein 90 (Hsp90), a prominent molecular chaperone, effectively limits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection but little is known about any interaction between Hsp90 and SARS-CoV-2 proteins. Here, we systematically analyzed the effects of the chaperone isoforms Hsp90α and Hsp90ß on individual SARS-CoV-2 viral proteins. Five SARS-CoV-2 proteins, namely nucleocapsid (N), membrane (M), and accessory proteins Orf3, Orf7a, and Orf7b were found to be novel clients of Hsp90ß in particular. Pharmacological inhibition of Hsp90 with 17-DMAG results in N protein proteasome-dependent degradation. Hsp90 depletion-induced N protein degradation is independent of CHIP, a ubiquitin E3 ligase previously identified for Hsp90 client proteins, but alleviated by FBXO10, an E3 ligase identified by subsequent siRNA screening. We also provide evidence that Hsp90 depletion may suppress SARS-CoV-2 assembly partially through induced M or N degradation. Additionally, we found that GSDMD-mediated pyroptotic cell death triggered by SARS-CoV-2 was mitigated by inhibition of Hsp90. These findings collectively highlight a beneficial role for targeting of Hsp90 during SARS-CoV-2 infection, directly inhibiting virion production and reducing inflammatory injury by preventing the pyroptosis that contributes to severe SARS-CoV-2 disease.

Targeting heat shock proteins 90 and 70: A promising remedy for both autoimmune bullous diseases and COVID-19.

Heat shock proteins (Hsps), including Hsp90 and Hsp70, are intra- and extracellular molecules implicated in cellular homeostasis and immune processes and are induced by cell stress such as inflammation and infection. Autoimmune bullous disorders (AIBDs) and COVID-19 represent potentially life-threatening inflammatory and infectious diseases, respectively. A significant portion of AIBDs remain refractory to currently available immunosuppressive therapies, which may represent a risk factor for COVID-19, and suffer from treatment side-effects. Despite advances in vaccination, there is still a need to develop new therapeutic approaches targeting SARS-CoV-2, especially considering vaccine hesitancy, logistical distribution challenges, and breakthrough infections. In this mini review, we briefly summarize the role of targeting Hsp90/70 as a promising double-edged sword in the therapy of AIBDs and COVID-19.

An Insight to Heat Shock Protein 90: A Remedy for Multiple Problems.

Heat shock protein 90 (Hsp90) is a chaperone protein that prevents many other proteins from aggregating by folding them in a certain way. Hsp90 consists of three structural domains: N-terminal, middle and C-terminal domains. Hsp90 has many activities in numerous proteins and signaling pathways like chimeric fusion proteins, steroid hormone receptors, tumor suppressor genes, and cell cycle regulatory proteins. The role of Hsp90 is not only in cancer but also in other diseases like COVID-19, leishmaniasis, diabetes, flavi virus, systemic sclerosis, grass carp reovirus, psoriasis, malaria, cardiac fibrosis, and alcohol-related liver diseases. This review is a compilation of the pharmacological profile of Hsp90 inhibitors, problems associated with them, and suggested remedies for the same.

Severe Acute Respiratory Syndrome Coronavirus 2 ORF8 Protein Inhibits Type I Interferon Production by Targeting HSP90B1 Signaling.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic that has currently infected over 430 million individuals worldwide. With the variant strains of SARS-CoV-2 emerging, a region of high mutation rates in ORF8 was identified during the early pandemic, which resulted in a mutation from leucine (L) to serine (S) at amino acid 84. A typical feature of ORF8 is the immune evasion by suppressing interferon response; however, the mechanisms by which the two variants of ORF8 antagonize the type I interferon (IFN-I) pathway have not yet been clearly investigated. Here, we reported that SARS-CoV-2 ORF8L and ORF8S with no difference inhibit the production of IFN-ß, MDA5, RIG-I, ISG15, ISG56, IRF3, and other IFN-related genes induced by poly(I:C). In addition, both ORF8L and ORF8S proteins were found to suppress the nuclear translocation of IRF3. Mechanistically, the SARS-CoV-2 ORF8 protein interacts with HSP90B1, which was later investigated to induce the production of IFN-ß and IRF3. Taken together, these results indicate that SARS-CoV-2 ORF8 antagonizes the RIG-I/MDA-5 signaling pathway by targeting HSP90B1, which subsequently exhibits an inhibitory effect on the production of IFN-I. These functions appeared not to be influenced by the genotypes of ORF8L and ORF8S. Our study provides an explanation for the antiviral immune suppression of SARS-CoV-2 and suggests implications for the pathogenic mechanism and treatment of COVID-19.

Binding of SARS-CoV-2 protein ORF9b to mitochondrial translocase TOM70 prevents its interaction with chaperone HSP90.

The emergence of the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a great threat to global health. ORF9b, an important accessory protein of SARS-CoV-2, plays a critical role in the viral host interaction, targeting TOM70, a member of the mitochondrial translocase of the outer membrane complex. The assembly between ORF9b and TOM70 is implicated in disrupting mitochondrial antiviral signaling, leading to immune evasion. We describe the expression, purification, and characterization of ORF9b alone or coexpressed with the cytosolic domain of human TOM70 in E. coli. ORF9b has 97 residues and was purified as a homodimer with an molecular mass of 22 kDa as determined by SEC-MALS. Circular dichroism experiments showed that Orf9b alone exhibits a random conformation. The ORF9b-TOM70 complex characterized by CD and differential scanning calorimetry showed that the complex is folded and more thermally stable than free TOM70, indicating strong binding. Importantly, protein-protein interaction assays demonstrated that full-length human Hsp90 is capable of binding to free TOM70 but not to the ORF9b-TOM70 complex. To narrow down the nature of this inhibition, the isolated C-terminal domain of Hsp90 was also tested. These results were used to build a model of the mechanism of inhibition, in which ORF9b efficiently targets two sites of interaction between TOM70 and Hsp90. The findings showed that ORF9b complexed with TOM70 prevents the interaction with Hsp90, and this is one major explanation for SARS-CoV-2 evasion of host innate immunity via the inhibition of the interferon activation pathway.

HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages

Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two innate immune cell types are activated by bitter products, including those secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells. NO also increases phagocytosis by macrophages. Using biochemistry and live-cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). Immunofluorescence showed that H441 cells express eNOS and T2Rs and that the bitter agonist denatonium benzoate activates NO production in a Ca2+- and HSP90-dependent manner in cells grown either as submerged cultures or at the air–liquid interface. In primary sinonasal epithelial cells, we determined that HSP90 inhibition reduces T2R-stimulated NO production and ciliary beating, which likely limits pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves as an innate immune modulator by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream Ca2+ signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases such as chronic rhinosinusitis, asthma, or cystic fibrosis.

HSP90 Inhibitors Modulate SARS-CoV-2 Spike Protein Subunit 1-Induced Human Pulmonary Microvascular Endothelial Activation and Barrier Dysfunction.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 5 million deaths worldwide. Multiple reports indicate that the endothelium is involved during SARS-Cov-2-related disease (COVID-19). Indeed, COVID-19 patients display increased thrombophilia with arterial and venous embolism and lung microcapillary thrombotic disease as major determinants of deaths. The pathophysiology of endothelial dysfunction in COVID-19 is not completely understood. We have investigated the role of subunit 1 of the SARS-CoV-2 spike protein (S1SP) in eliciting endothelial barrier dysfunction, characterized dose and time relationships, and tested the hypothesis that heat shock protein 90 (HSP90) inhibitors would prevent and repair such injury. S1SP activated (phosphorylated) IKBα, STAT3, and AKT and reduced the expression of intercellular junctional proteins, occludin, and VE-cadherin. HSP90 inhibitors (AT13387 and AUY-922) prevented endothelial barrier dysfunction and hyperpermeability and reduced IKBα and AKT activation. These two inhibitors also blocked S1SP-mediated barrier dysfunction and loss of VE-cadherin. These data suggest that spike protein subunit 1 can elicit, by itself, direct injury to the endothelium and suggest a role of HSP90 inhibitors in preserving endothelial functionality.

A Comparative Transcriptomic Analysis Reveals That HSP90AB1 Is Involved in the Immune and Inflammatory Responses to Porcine Deltacoronavirus Infection.

PDCoV is an emerging enteropathogenic coronavirus that mainly causes acute diarrhea in piglets, seriously affecting pig breeding industries worldwide. To date, the molecular mechanisms of PDCoV-induced immune and inflammatory responses or host responses in LLC-PK cells in vitro are not well understood. HSP90 plays important roles in various viral infections. In this study, HSP90AB1 knockout cells (HSP90AB1KO) were constructed and a comparative transcriptomic analysis between PDCoV-infected HSP90AB1WT and HSP90AB1KO cells was conducted using RNA sequencing to explore the effect of HSP90AB1 on PDCoV infection. A total of 1295 and 3746 differentially expressed genes (DEGs) were identified in PDCoV-infected HSP90AB1WT and HSP90AB1KO cells, respectively. Moreover, most of the significantly enriched pathways were related to immune and inflammatory response-associated pathways upon PDCoV infection. The DEGs enriched in NF-κB pathways were specifically detected in HSP90AB1WT cells, and NF-κB inhibitors JSH-23, SC75741 and QNZ treatment reduced PDCoV infection. Further research revealed most cytokines associated with immune and inflammatory responses were upregulated during PDCoV infection. Knockout of HSP90AB1 altered the upregulated levels of some cytokines. Taken together, our findings provide new insights into the host response to PDCoV infection from the transcriptome perspective, which will contribute to illustrating the molecular basis of the interaction between PDCoV and HSP90AB1.

Morphological Alterations and Stress Protein Variations in Lung Biopsies Obtained from Autopsies of COVID-19 Subjects.

Molecular chaperones, many of which are heat shock proteins, play a role in cell stress response and regulate the immune system in various ways, such as in inflammatory/autoimmune reactions. It would be interesting to study the involvement of these molecules in the damage done to COVID-19-infected lungs. In our study, we performed a histological analysis and an immunomorphological evaluation on lung samples from subjects who succumbed to COVID-19 and subjects who died from other causes. We also assessed Hsp60 and Hsp90 distribution in lung samples to determine their location and post-translational modifications. We found histological alterations that could be considered pathognomonic for COVID-19-related lung disease. Hsp60 and Hsp90 immunopositivity was significantly higher in the COVID-19 group compared to the controls, and immunolocalization was in the plasma membrane of the endothelial cells in COVID-19 subjects. The colocalization ratios for Hsp60/3-nitrotyrosine and Hsp60/acetylate-lisine were significantly increased in the COVID-19 group compared to the control group, similar to the colocalization ratio for Hsp90/acetylate-lisine. The histological and immunohistochemical findings led us to hypothesize that Hsp60 and Hsp90 might have a role in the onset of the thromboembolic phenomena that lead to death in a limited number of subjects affected by COVID-19. Further studies on a larger number of samples obtained from autopsies would allow to confirm these data as well as discover new biomarkers useful in the battle against this disease.

Role of Heat Shock Proteins (HSP70 and HSP90) in Viral Infection.

Heat shock proteins (HSPs) are a large group of chaperones found in most eukaryotes and bacteria. They are responsible for the correct protein folding, protection of the cell against stressors, presenting immune and inflammatory cytokines; furthermore, they are important factors in regulating cell differentiation, survival and death. Although the biological function of HSPs is to maintain cell homeostasis, some of them can be used by viruses both to fold their proteins and increase the chances of survival in unfavorable host conditions. Folding viral proteins as well as replicating many different viruses are carried out by, among others, proteins from the HSP70 and HSP90 families. In some cases, the HSP70 family proteins directly interact with viral polymerase to enhance viral replication or they can facilitate the formation of a viral replication complex and/or maintain the stability of complex proteins. It is known that HSP90 is important for the expression of viral genes at both the transcriptional and the translational levels. Both of these HSPs can form a complex with HSP90 and, consequently, facilitate the entry of the virus into the cell. Current studies have shown the biological significance of HSPs in the course of infection SARS-CoV-2. A comprehensive understanding of chaperone use during viral infection will provide new insight into viral replication mechanisms and therapeutic potential. The aim of this study is to describe the molecular basis of HSP70 and HSP90 participation in some viral infections and the potential use of these proteins in antiviral therapy.

Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.

Drug-repurposing against COVID-19 by targeting a key signaling pathway: An in silico study.

Currently, a plethora of information has been accumulated concerning COVID-19, including the transmission pathway of SARs-CoV-2. Thus, we retrieved targets associated with the development of COVID-19 via PubChem. A total of 517 targets were identified, and signaling pathways responded after infection of SARs-CoV-2 in humans constructed a bubble chart using RPackage. The bubble chart result suggested that the key signaling pathway against COVID-19 was the estrogen signaling pathway associated with AKT1, HSP90AB1, BCL2 targets. The three targets have the strongest affinity with three ligands-Akti-1/2, HSP990, S55746, respectively. In conclusion, this work provides three key elements to alleviate COVID-19 symptoms might be anti-inflammatory effects on SARs-CoV-2-infected lung cells.

Global analysis of protein-RNA interactions in SARS-CoV-2-infected cells reveals key regulators of infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs, and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Among them are several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

First Virtual International Congress on Cellular and Organismal Stress Responses, November 5-6, 2020.

Members of the Cell Stress Society International (CSSI), Patricija van Oosten-Hawle (University of Leeds, UK), Mehdi Mollapour (SUNY Upstate Medical University, USA), Andrew Truman (University of North Carolina at Charlotte, USA) organized a new virtual meeting format which took place on November 5-6, 2020. The goal of this congress was to provide an international platform for scientists to exchange data and ideas among the Cell Stress and Chaperones community during the Covid-19 pandemic. Here we will highlight the summary of the meeting and acknowledge those who were honored by the CSSI.

Are Hsp90 inhibitors good candidates against Covid-19?

Drug reposition, or repurposing, has become a promising strategy in therapeutics due to its advantages in several aspects of drug therapy. General drug development is expensive and can take more than 10 years to go through the designing, development, and necessary approval steps. However, established drugs have already overcome these steps and thus a potential candidate may be already available decreasing the risks and costs involved. Viruses invade cells, usually provoking biochemical changes, leading to tissue damage, alteration of normal physiological condition in organisms and can even result in death. Inside the cell, the virus finds the machinery necessary for its multiplication, as for instance the protein quality control system, which involves chaperones and Hsps (heat shock proteins) that, in addition to physiological functions, help in the stabilization of viral proteins. Recently, many inhibitors of Hsp90 have been developed as therapeutic strategies against diseases such as the Hsp90 inhibitors used in anticancer therapy. Several shreds of evidence indicate that these inhibitors can also be used as therapeutic strategies against viruses. Therefore, since a drug treatment for COVID-19 is urgently needed, this review aims to discuss the potential use of Hsp90 inhibitors in the treatment of this globally threatening disease.