Medium-Throughput Detection of Hsp90/Cdc37 Protein-Protein Interaction Inhibitors Using a Split Renilla Luciferase-Based Assay

The protein-folding chaperone Hsp90 enables the maturation and stability of various oncogenic signaling proteins and is thus pursued as a cancer drug target. Folding in particular of protein kinases is assisted by the co-chaperone Cdc37. Several inhibitors against the Hsp90 ATP-binding site have been developed. However, they displayed significant toxicity in clinical trials. By contrast, the natural product conglobatin A has an exceptionally low toxicity in mice. It targets the protein-protein interface (PPI) of Hsp90 and Cdc37, suggesting that interface inhibitors have an interesting drug development potential. In order to identify inhibitors of the Hsp90/Cdc37 PPI, we have established a mammalian cell lysate-based, medium-throughput amenable split Renilla luciferase assay. This assay employs N-terminal and C-terminal fragments of Renilla luciferase fused to full-length human Hsp90 and Cdc37, respectively. We expect that our assay will allow for the identification of novel Hsp90/Cdc37 interaction inhibitors. Such tool compounds will help to evaluate whether the toxicity profile of Hsp90/Cdc37 PPI inhibitors is in general more favorable than that of ATP-competitive Hsp90 inhibitors. Further development of such tool compounds may lead to new classes of Hsp90 inhibitors with applications in cancer and other diseases

Multiomics integrative analysis reveals antagonistic roles of CBX2 and CBX7 in metabolic reprogramming of breast cancer

Striking similarity exists between metabolic changes associated with embryogenesis and tumorigenesis. Chromobox proteins-CBX2/4/6/7/8, core components of canonical polycomb repressor complex 1, play essential roles in embryonic development and aberrantly expressed in breast cancer. Understanding how altered CBX expression relates to metabolic reprogramming in breast cancer may reveal vulnerabilities of therapeutic pertinence. Using transcriptomic and metabolomic data from breast cancer patients (N > 3000 combined), we performed pathway-based analysis and identified outstanding roles of CBX2 and CBX7 in positive and negative regulation of glucose metabolism, respectively. Genetic ablation experiments validated the contrasting roles of two isoforms in cancer metabolism and cell growth. Furthermore, we provide evidence for the role of mammalian target of rapamycin complex 1 signaling in mediating contrary effects of CBX2 and CBX7 on breast cancer metabolism. Underpinning the biological significance of metabolic roles, CBX2 and CBX7 were found to be the most up- and downregulated isoforms, respectively, in breast tumors compared with normal tissues. Moreover, CBX2 and CBX7 expression (not other isoforms) correlated strongly, but oppositely, with breast tumor subtype aggressiveness and the proliferation markers. Consistently, genomic data also showed higher amplification frequency of CBX2, not CBX7, in breast tumors. Highlighting the clinical significance of findings, disease-specific survival and drug sensitivity analysis revealed that CBX2 and CBX7 predicted patient outcome and sensitivity to FDA-approved/investigational drugs. In summary, this work identifies novel cross talk between CBX2/7 and breast tumor metabolism, and the results presented may have implications in strategies targeting breast cancer

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

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

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

Simple SummaryThe correct folding of proteins is essential for their activity. Therefore, cells have evolved protein-folding chaperones, such as Hsp90. Interestingly, in several cancer cells, Hsp90 appears to have a role that is more important than normal. The current working model suggests that, with the help of its co-chaperone, Cdc37, it stabilizes mutant kinases. However, Hsp90, together with Cdc37, assists additional proteins that may be relevant in cancer. We demonstrate that the Hsp90-dependent stability of the transcription factor HIF-1 alpha and one of its downstream transcriptional targets, galectin-3, is important to maintain the elevated activity of the major oncogene KRAS. This is because galectin-3 stabilizes the MAPK-signaling complexes of K-Ras, which is called a nanocluster. In addition, we identified six drug-like small molecules that inhibit the Hsp90/Cdc37 protein interface at low micro molar concentrations. Given the co-occurrence of mutant KRAS with high HIF-1 alpha and high galectin-3 levels in pancreatic cancer, our results suggest an application of Hsp90 inhibitors in this cancer type.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-1 alpha. This mechanism suggests a higher drug sensitivity in the context of KRAS mutant, HIF-1 alpha-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 mu M-44 mu M 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

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Development of Novel Drugs Targeting Chaperones of Oncogenic K-Ras

KRAS mutations account globally for about one million deaths per year, but there is as yet no approved drug against KRAS. K-Ras proteins are organized into di-/oligomeric nanoscale signaling complexes, known as nanoclusters on the plasma membrane. In this thesis, I describe two targeting approaches to indirectly inhibit the oncogenic activity of K-Ras. First, we designed inhibitors that block the interaction between the trafficking chaperone PDE6D and K-Ras, thus blocking membrane localization of K-Ras. Secondly, we showed how the major folding chaperone Hsp90 and its co-chaperone Cdc37 affect K-Ras signaling. We then went on to develop and test novel inhibitors against the interface between Hsp90 and Cdc37. K-Ras requires several post-translational modifications, such as farnesylation, for proper organization on the plasma membrane. Interaction of farnesylated KRas with the trafficking chaperone PDE6D results in its effective solubilization and translocation in the cytosol. Available PDE6D inhibitors are easily dislodged from PDE6D through the GTP-ARL2 unloading mechanism, consequently limiting the overall cellular potency of these inhibitors. To address this problem, we improved the compound design to withstand the ejection mechanism and added a cell penetration group to increase the bioavailability. The second part connects to our previous finding, showing that the inhibition of Hsp90/Cdc37 by conglobatin A selectively blocks the activity of K-Ras but not H-Ras, and inhibits stemness activity of cancer cells. However, the exact mechanism by which Hsp90/Cdc37 inhibition blocks the activity of K-Ras remained unclear. Here, we discovered that the inhibition of Hsp90 downregulates the K-Ras nanocluster modulator galectin-3 by inhibiting HIF-1α. Decreased expression levels of galectin-3 and Hsp90-clients B- and C-Raf jointly contributed to selectively disrupt K-Ras membrane nanoclusters, thus blocking the oncogenic activity of K-Ras. In order to identify novel Hsp90/Cdc37 inhibitors, we first selected compounds from a computational screening and then validated their ability to interrupt the Hsp90/Cdc37 complex in a split Renilla luciferase assay. Finally, we discovered two compounds that inhibited the Hsp90/Cdc37 complex formation. By assessing these compounds in cellular assays, we confirmed their K-Ras membrane organization disrupting activity and the impairment of the signaling pathways downstream of K-Ras. Furthermore, these compounds also decreased K-Ras dependent cancer cell proliferation in 2D monolayers, 3D spheroid growth and microtumor formation. Taken together, the work of this thesis has led to the development and characterization of novel small molecule inhibitors that indirectly target K-Ras. Our findings may form the basis for the development of future therapeutic agents against K-Ras dependent human diseases. ---------- KRAS-mutationer står globalt för cirka en miljon dödsfall per år, men det finns ännu inget godkänt läkemedel mot KRAS. K-Ras-proteiner är organiserade i di- /oligomera signalkomplex i nanoskala, kända som nanokluster på plasmamembranet. I denna avhandling beskriver jag två olika metoder för att indirekt hämma den onkogena aktiviteten hos K-Ras. Först designade vi hämmare som blockerar interaktionen mellan den intracellulära transport chaperonen PDE6D och K-Ras, för att blockera membranlokalisering av K-Ras. Som andra visade vi hur den huvudsakliga proteinvecknings chaperonen Hsp90 och dess co-chaperon Cdc37 påverkar K-Ras-signalering. Vi fortsatte sedan med att utveckla och testa nya hämmare mot samspelmellan Hsp90 och Cdc37. För korrekt organisering på plasmamembranen kräver K-Ras flera posttranslationell modifieringar, såsom farnesylering. Interaktionen mellan den farnesylerade K-Ras och chaperonen PDE6D resulterar i effektiv nedbrytning av K-Ras och dess translokering i cytosolen. Tillgängliga PDE6D-hämmare lösgörs lätt från PDE6D genom en GTP-ARL2- avstötningsmekanism. Detta har till följd att den totala cellulära effekten hos dessa hämmare begränsas. För att lösa detta problem förbättrade vi föreningens (hämmarens) design, så att den bättre kan motstå avstötningsmekanismen. Dessutom ökade vi dess biologiska tillgänglighet genom att lägga till en kemisk grupp, som ökar hämmarens förmåga att penetrera cellmembranen. Den andra delen ansluter till våra tidigare forskningsresultat som visar att blockering av Hsp90/Ccd37 med conglobatin A blockerar selektivt K-Ras aktivitet utan att påverka H-Ras aktivitet, samtidigt som den inhiberar stamcellsförmågan i cancerceller. Den exakta mekanismen genom vilken Hsp90 / Cdc37-inhibering blockerar K-Ras aktivitet förblev emellertid oklar. I denna avhandling upptäckte vi att hämningen av Hsp90 nedreglerar galectin-3, en nanoklustermodulator för K-Ras, genom att hämma HIF-lα. Minskade expressionsnivåer av galectin-3 och Hsp90-klienterna B- och C-Raf bidrog gemensamt till att selektivt hindra K-Ras från att bilda membran-nanokluster, som i sin tur ledde till att den onkogena aktiviteten hos K-Ras blockerades. För att identifiera nya Hsp90 / Cdc37-hämmare valde vi först ut föreningar genom datorscreening och validerade sedan deras förmåga att avbryta Hsp90 /Cdc37-komplexet med hälp av ett delat Renilla-luciferastest. Vi upptäckte slutligen två föreningar som hämmade komplexbildningen av Hsp90 / Cdc37. Genom att utvärdera dessa föreningar i cellulära analyser, bekräftade vi deras förmåga att hindra K-Ras-membranorganisation och försämring av signalvägarna nedströms från K-Ras. Dessa föreningar minskade dessutom också K-Rasberoende tillväxt av cancerceller i 2D-monolager, 3D-sfäroid tillväxt och mikrotumörbildning. Sammantaget har arbetet i denna avhandling lett till utveckling och karakterisering av nya småmolekylära hämmare som indirekt riktar sig mot K-Ras. Våra resultat kan ligga till grund för utvecklingen av framtida terapeutiska medel mot K-Ras-beroende sjukdomar