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In silico methods alongisde dibromomaleimide stapling scanning and sequence hybridization were used to inform the design of BCL-xL selective peptideomimetic ligands.

The ubiquitously expressed glucocorticoid receptor (GR) is a nuclear receptor that controls a broad range of biological processes and is activated by steroidal glucocorticoids such as hydrocortisone or dexamethasone. Glucocorticoids are used to treat a wide variety of conditions, from inflammation to cancer but suffer from a range of side effects that motivate the search for safer GR modulators. GR is also regulated outside the steroid-binding site through protein-protein interactions (PPIs) with 14-3-3 adapter proteins. Manipulation of these PPIs will provide insights into noncanonical GR signaling as well as a new level of control over GR activity. We report the first molecular glues that selectively stabilize the 14-3-3/GR PPI using the related nuclear receptor estrogen receptor α (ERα) as a selectivity target to drive design. These 14-3-3/GR PPI stabilizers can be used to dissect noncanonical GR signaling and enable the development of novel atypical GR modulators.

Understanding how microbiota evade and suppress host immunity is critical to our knowledge of how beneficial microbes persist in association with a host. Prior work has shown that secretion of organic acids by beneficial microbes is sufficient to suppress plant immunity.

Supplementary Data from Aprataxin Tumor Levels Predict Response of Colorectal Cancer Patients to Irinotecan-based Treatment

Purpose: Irinotecan (CPT11) treatment significantly improves the survival of colorectal cancer patients and is routinely used for the treatment of these patients, alone or in combination with other agents. However, only 20% to 30% of patients show an objective response to irinotecan, and there is great need for new molecular markers capable of identifying the subset of patients who are unlikely to respond.Experimental Design: Here we used microarray analysis of a panel of 30 colorectal cancer cell lines and immunohistochemistry to identify and validate a new biomarker of response to irinotecan.Results: A good correlation was observed between irinotecan sensitivity and the expression of aprataxin (APTX), a histidine triad domain superfamily protein involved in DNA repair. Moreover, using an isogenic in vitro system deficient in APTX, we show that aprataxin directly regulates the cellular sensitivity to camptothecin, suggesting that it could be used to predict patient response to irinotecan. We constructed a tissue microarray containing duplicate tumor samples from 135 patients that received irinotecan for the treatment of advanced colorectal cancer. Immunohistochemical assessment of the tumor levels of aprataxin showed a significant association with treatment response and patient survival. Patients with low aprataxin had longer progression-free (9.2 versus 5.5 months; P = 0.03) and overall survival (36.7 versus 19.0 months; P = 0.008) than patients with high tumor aprataxin. No associations were found between coding APTX variants and aprataxin levels or camptothecin sensitivity.Conclusions: These results show that aprataxin tumor levels can be used to identify patients with low probability of response to irinotecan-based therapy who are ideal candidates to receive treatment with alternative agents in an attempt to improve patient survival. Clin Cancer Res; 16(8); 2375–82. ©2010 AACR.

Supplementary Figures 1-5 from PIK3CA Mutation/PTEN Expression Status Predicts Response of Colon Cancer Cells to the Epidermal Growth Factor Receptor Inhibitor Cetuximab

Supplementary Figures 1-6, Tables 1-6 from The Receptor Tyrosine Kinase EPHB4 Has Tumor Suppressor Activities in Intestinal Tumorigenesis

A, quantification of western blot analysis for phosphorylation of MEK and ERK after treatment with decitabine for six days in a KRAS-mutant (MCAS) and KRAS-wild type (RMGI) ovarian cancer cell line as depicted in the main text. B, phosphorylation of MEK and ERK was probed by western blot after treatment with decitabine for six days in a KRAS-mutant ovarian cancer cell line (OVCAR8). Quantification was performed using Image J. C, KRAS-mutant ovarian cancer cell line, OVCAR8, was co-treated with decitabine and navitoclax for six days (nine replicates, mean). Data were normalized to the ATP levels (CTG) measured for the corresponding DMSO-treated controls (6 days) to illustrate the overall decrease in cell viability for the combination treatment. Data is a representative example of two independent experiments. D, combined bisulfite restriction analysis of the transcriptional start site of BNIP3 in MCAS cell line treated with decitabine (0.5 μM) or DMSO for six days.

Supplementary Table 4 from Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

The differential mutual information score (Ic, methods) is used in this table to correlate pathways with activity of decitabine in solid tumor cell lines.

The differential mutual information score (Ic, methods) is used in this table to correlate gene mutations and copy number variations with activity of decitabine in solid tumor cell lines.

Supplementary Figures 1-8 from Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

PDF - 404K, Supplementary Figure 1. TR3 expression in normal and neoplastic ovarian epithelium. Supplementary Figure 2. TR3 expression in ovarian cancer epithelial sub-types. Supplementary Figure 3. Association of TR3 expression with clinical data in publically available databases. Supplementary Figure 4. Effects of cisplatin in normal human ovarian surface epithelial cells (HOSE). Supplementary Figure 5. Effects of cisplatin in A2780 PAR and CP20 ovarian cancer cells. Supplementary Figure 6. Effect of cisplatin on cell growth and cytotoxicity in OVCAR8 ShScr and ShTR3 cells. Supplementary Figure 7. Linkage of JNK and Akt regulation of TR3 subcellular localization to apoptotic effects of cisplatin.

Supplementary Materials, Tables 1-4 from EPHB4 and Survival of Colorectal Cancer Patients

Supplementary Figure 1 from EPHB4 and Survival of Colorectal Cancer Patients

Supplementary Table 5 from Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

PDF - 80K, Additional details about methodology used for Immunofluorescence, Immunohistochemistry, Western Blotting, quantitative RT-PCR and case selection and TMA generation.

Supplementary Table 1-3 from Mechanisms of Inactivation of the Receptor Tyrosine Kinase EPHB2 in Colorectal Tumors

Supplementary Tables 1-3 from Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

Supplementary Methods and Materials from Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

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Cetuximab is a monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). Although approved for use in EGFR-overexpressing advanced colorectal cancer, recent studies have shown a lack of association between EGFR overexpression and cetuximab response, requiring the identification of novel biomarkers predictive of response to this agent. To do so, 22 colon cancer cell lines were screened for cetuximab response in vitro and sensitive and resistant lines were identified. In sensitive cell lines, cetuximab induced a G0-G1 arrest without inducing apoptosis. Notably, cetuximab-sensitive but not cetuximab-resistant cell lines were preferentially responsive to EGF-stimulated growth. Whereas neither EGFR protein/mRNA expression nor gene copy number correlated with cetuximab response, examination of the mutation status of signaling components downstream of EGFR showed that cell lines with activating PIK3CA mutations or loss of PTEN expression (PTEN null) were more resistant to cetuximab than PIK3CA wild type (WT)/PTEN-expressing cell lines (14 ± 5.0% versus 38.5 ± 6.4% growth inhibition, mean ± SE; P = 0.008). Consistently, PIK3CA mutant isogenic HCT116 cells showed increased resistance to cetuximab compared with PIK3CA WT controls. Furthermore, cell lines that were PIK3CA mutant/PTEN null and Ras/BRAF mutant were highly resistant to cetuximab compared with those without dual mutations/PTEN loss (10.8 ± 4.3% versus 38.8 ± 5.9% growth inhibition, respectively; P = 0.002), indicating that constitutive and simultaneous activation of the Ras and PIK3CA pathways confers maximal resistance to this agent. A priori screening of colon tumors for PTEN expression status and PIK3CA and Ras/BRAF mutation status could help stratify patients likely to benefit from this therapy. [Cancer Res 2008;68(6):1953–60]

The receptor tyrosine kinase EPHB2 has recently been shown to be a direct transcriptional target of TCF/β-catenin. Premalignant lesions of the colon express high levels of EPHB2 but the expression of this kinase is reduced or lost in most colorectal carcinomas. In addition, inactivation of EPHB2 has been shown to accelerate tumorigenesis initiated by APC mutation in the colon and rectum. In this study, we investigated the molecular mechanisms responsible for the inactivation of EPHB2 in colorectal tumors. We show here the presence of mutations in repetitive sequences in exon 17 of EPHB2 in 6 of 29 adenomas with microsatellite instability (MSI), and 101 of 246 MSI carcinomas (21% and 41%, respectively). Moreover, we found EPHB2 promoter hypermethylation in 54 of the 101 colorectal tumors studied (53%). Importantly, EPHB2 expression was restored after treatment of EPHB2-methylated colon cancer cells with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine. In conclusion, in this study, we elucidate the molecular mechanisms of inactivation of EPHB2 and show for the first time the high incidence of frameshift mutations in MSI colorectal tumors and aberrant methylation of the regulatory sequences of this important tumor suppressor gene.

WEAVE, the new wide-field, massively multiplexed spectroscopic survey facility for the William Herschel Telescope, will see first light in late 2022. WEAVE comprises a new 2-degree field-of-view prime-focus corrector system, a nearly 1000-multiplex fibre positioner, 20 individually deployable 'mini' integral field units (IFUs), and a single large IFU. These fibre systems feed a dual-beam spectrograph covering the wavelength range 366$-$959\,nm at $R\sim5000$, or two shorter ranges at $R\sim20\,000$. After summarising the design and implementation of WEAVE and its data systems, we present the organisation, science drivers and design of a five- to seven-year programme of eight individual surveys to: (i) study our Galaxy's origins by completing Gaia's phase-space information, providing metallicities to its limiting magnitude for $\sim$3 million stars and detailed abundances for $\sim1.5$ million brighter field and open-cluster stars; (ii) survey $\sim0.4$ million Galactic-plane OBA stars, young stellar objects and nearby gas to understand the evolution of young stars and their environments; (iii) perform an extensive spectral survey of white dwarfs; (iv) survey $\sim400$ neutral-hydrogen-selected galaxies with the IFUs; (v) study properties and kinematics of stellar populations and ionised gas in $z1$ million spectra of LOFAR-selected radio sources; (viii) trace structures using intergalactic/circumgalactic gas at $z>2$. Finally, we describe the WEAVE Operational Rehearsals using the WEAVE Simulator., 41 pages, 27 figures, accepted for publication by MNRAS

Highlights • Panobinostat in combination with olaparib reduces homologous recombination (HR) proficient tumor cell viability. • This combination favors anti-tumorigenic macrophages and enhances CD8+ T cell infiltration into tumors. • As a result, this combination targets HR-proficient ovarian tumors directly and through the tumor microenvironment., Histone deacetylase inhibitors (HDACi) sensitize homologous recombination (HR)-proficient human ovarian cancer cells to PARP inhibitors (PARPi). To investigate mechanisms of anti-tumor effects of combined HDACi/PARPi treatment we performed transcriptome analysis in HR- proficient human ovarian cancer cells and tested drug effects in established immunocompetent mouse ovarian cancer models. Human SKOV-3 cells were treated with vehicle (Con), olaparib (Ola), panobinostat (Pano) or Pano+Ola and RNA-seq analysis performed. DESeq2 identified differentially expressed HR repair and immune transcripts. Luciferised syngeneic mouse ovarian cancer cells (ID8-luc) were treated with the HDACi panobinostat alone or combined with olaparib and effects on cell viability, apoptosis, DNA damage and HR efficiency determined. C57BL/6 mice with intraperitoneally injected ID8-luc cells were treated with panobinostat and/or olaparib followed by assessment of tumor burden, markers of cell proliferation, apoptosis and DNA damage, tumor-infiltrating T cells and macrophages, and other immune cell populations in ascites fluid. There was a significant reduction in expression of 20/37 HR pathway genes by Pano+Ola, with immune and inflammatory-related pathways also significantly enriched by the combination. In ID8 cells, Pano+Ola decreased cell viability, HR repair, and enhanced DNA damage. Pano+Ola also co-operatively reduced tumor burden and proliferation, increased tumor apoptosis and DNA damage, enhanced infiltration of CD8+ T cells into tumors, and decreased expression of M2-like macrophage markers. In conclusion, panobinostat in combination with olaparib targets ovarian tumors through both direct cytotoxic and indirect immune-modulating effects.

Objective Poly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer. Methods We assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model. Results Entinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability. Conclusion Entinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.

Under the shielding effect of nanomicelles, a sustainable micellar technology for the design and convenient synthesis of ligand-free oxidizable ultrasmall Pd(0) nanoparticles (NPs) and their subsequent catalytic exploration for couplings of water-sensitive acid chlorides in water is reported. A proline-derived amphiphile, PS-750-M, plays a crucial role in stabilizing these NPs, preventing their aggregation and oxidation state changes. These NPs were characterized using 13C nuclear magnetic resonance (NMR), infrared (IR), and surface-enhanced Raman scattering (SERS) spectroscopy to evaluate the carbonyl interactions of PS-750-M with Pd. High-resolution transmission electron microscopy (HRTEM) and energy-dispersive X-ray spectroscopy (EDX) studies were performed to reveal the morphology, particle size distribution, and chemical composition, whereas X-ray photoelectron spectroscopy (XPS) measurements unveiled the oxidation state of the metal. In the cross-couplings of water-sensitive acid chlorides with boronic acids, the micelle’s shielding effect and boronic acids plays a vital role in preventing unwanted side reactions, including the hydrolysis of acid chlorides under basic pH. This approach is scalable and the applications are showcased in multigram scale reactions.

The understanding and rational design of heterogeneous catalysts for complex reactions, such as CO2 reduction, requires knowledge of elementary steps and chemical species prevalent on the catalyst surface under operating conditions. Using in situ nanoscale surface-enhanced Raman scattering, we probe the surface of a Ag nanoparticle during plasmon-excitation-driven CO2 reduction in water. Enabled by the high spatiotemporal resolution and surface sensitivity of our method, we detect a rich array of C1–C4 species formed on the photocatalytically active surface. The abundance of multi-carbon compounds, such as butanol, suggests the favorability of kinetically challenging C–C coupling on the photoexcited Ag surface. Another advance of this work is the use of isotope labeling in nanoscale probing, which allows confirmation that detected species are the intermediates and products of the catalytic reaction rather than spurious contaminants. The surface chemical knowledge made accessible by our approach will inform the modeling and engineering of catalysts. In situ vibrational spectroscopy affords a powerful tool for probing elementary chemical processes on catalytic surfaces. Using surface enhanced Raman scattering, authors identify an array of multicarbon species formed on a Ag nanoparticle catalyst in plasmon-driven reduction of CO2 in water.

Protein-protein interactions (PPIs) intimately govern various biological processes and disease states and therefore have been identified as attractive therapeutic targets for small-molecule drug discovery. However, the development of highly potent inhibitors for PPIs has proven to be extremely challenging with limited clinical success stories. Herein, we report irreversible inhibitors of the human double minute 2 (HDM2)/p53 PPI, which employ a reactive N-acyl-N-alkyl sulfonamide (NASA) group as a warhead. Mass-based analysis successfully revealed the kinetics of covalent inhibition and the modification sites on HDM2 to be the N-terminal α-amine and Tyr67, both rarely seen in traditional covalent inhibitors. Finally, we demonstrated prolonged p53-pathway activation and more effective induction of the p53-mediated cell death in comparison to a noncovalent inhibitor. This study highlights the potential of the NASA warhead as a versatile electrophile for the covalent inhibition of PPIs and opens new avenues for the rational design of potent covalent PPI inhibitors.

Background: Ovarian cancer is the most lethal gynecologic malignancy, with a 5-year survival rate of less than 50%. To have a major impact on improving clinical outcomes, a better understanding of the progression of disease and identification of truly novel treatment strategies is critical. Canonical nuclear factor-kappaB (NF-κB) signaling has been extensively studied in preclinical ovarian cancer models; however, systemic NF-κB inhibitors have had disappointing results in early clinical trials. Whether the alternative, non-canonical NF-κB signaling pathway (nc-NF-κB) represents a distinct and improved target for therapy in ovarian cancer is unknown. Here, we aim to investigate the role played by nc-NF-κB signaling both in malignant epithelial cells and in macrophages, a key immune cell component in the tumor microenvironment. Repolarizing tumor-associated macrophages (TAMs) away from pro-tumor M2-like functions towards an anti-tumor M1-like phenotype is a critical priority in macrophage-directed therapies. Methods: In ovarian cancer cell lines, mouse ovarian TAMs cultured ex vivo, and immortalized bone marrow-derived macrophages (BMDMs), nuclear levels of p52 were measured by western blot as a read-out of active nc-NF-κB signaling. Effects of macrophage polarization on nc-NF-κB signaling were determined in unpolarized (M0), M1 polarized (LPS/IFNγ treated) or M2 polarized (IL-4 treated) BMDMs. Finally, effects of inhibiting nc-NF-κB using a specific peptide inhibitor of p52 nuclear import (SN52) on cancer cell growth and macrophage phenotype were determined in sulforhodamine B growth assays and by western blot analysis of PCNA (proliferation marker) and arginase-1 (M2 macrophage marker). Results: We observed high levels of p52 expression in the majority of ovarian cancer cell lines, and in our cultured macrophage models. The functional relevance of these observations was confirmed by experiments showing reduced growth in ovarian cancer cells treated with SN52 and reduced expression of arginase-1 in SN52-treated macrophages. We saw similar results in breast cancer cells and mouse mammary TAMs, showing that nc-NF-κB signaling may also have pro-tumor effects in other cancer types. Complementary studies showed that M2 polarized macrophages expressed markedly higher levels of nuclear p52 than observed in M0 or M1 cells. Based on these promising results, experiments (i) using transgenic mice to allow targeted, inducible activation of nc-NF-κB in macrophages during ovarian cancer progression and (ii) testing the therapeutic effects of SN52 in these cancer models are underway. Conclusions: Although further studies are required to establish the translational relevance of our findings, we hope to identify non-canonical NF-κB signaling as a new therapeutic target in malignant epithelium and macrophages in ovarian cancer and potentially other cancer types. Citation Format: Andrew J. Wilson, Dominique Parker, Robert Cheng, Todd Giorgio, Marta Crispens, Fiona Yull. Investigating the role of non-canonical NF-kappaB signaling in tumor cells and macrophages in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4973.

Abstract: The BCL‐2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL‐2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non‐antibody‐binding proteins based on a conserved scaffold) to identify ligands for MCL‐1, BCL‐xL, BCL‐2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL‐2 protein. For anti‐apoptotic targets BCL‐xL and MCL‐1, competitive inhibition of their canonical protein‐protein interactions is demonstrated. Co‐crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug‐bound‐like conformation. These proof‐of‐concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL‐2 family modulators and more generally other protein‐protein interaction inhibitors., Affimer reagents (non‐antibody binding proteins) are identified that bind selectively to the anti‐apoptotic proteins BCL‐xL and MCL‐1. Crystallographic studies reveal an unprecedented mode of molecular recognition in which flexible loops from the Affimer dock into the BH3 binding cleft as opposed to a canonical α‐helix. Furthermore the Affimers induce changes in the target proteins towards desirable drug‐bound‐like conformations.

Background New treatment options for ovarian cancer are urgently required. Tumor-associated macrophages (TAMs) are an attractive target for therapy; repolarizing TAMs from M2 (pro-tumor) to M1 (anti-tumor) phenotypes represents an important therapeutic goal. We have previously shown that upregulated NF-kappaB (NF-κB) signaling in macrophages promotes M1 polarization, but effects in the context of ovarian cancer are unknown. Therefore, we aimed to investigate the therapeutic potential of increasing macrophage NF-κB activity in immunocompetent mouse models of ovarian cancer. Methods We have generated a transgenic mouse model, termed IKFM, which allows doxycycline-inducible overexpression of a constitutively active form of IKK2 (cIKK2) specifically within macrophages. The IKFM model was used to evaluate effects of increasing macrophage NF-κB activity in syngeneic murine TBR5 and ID8-Luc models of ovarian cancer in two temporal windows: 1) in established tumors, and 2) during tumor implantation and early tumor growth. Tumor weight, ascites volume, ascites supernatant and cells, and solid tumor were collected at sacrifice. Populations of macrophages and T cells within solid tumor and/or ascites were analyzed by immunofluorescent staining and qPCR, and soluble factors in ascitic fluid were analyzed by ELISA. Comparisons of control versus IKFM groups were performed by 2-tailed Mann-Whitney test, and a P-value Results Increased expression of the cIKK2 transgene in TAMs from IKFM mice was confirmed at the mRNA and protein levels. Tumors from IKFM mice, regardless of the timing of doxycycline (dox) administration, demonstrated greater necrosis and immune infiltration than control tumors. Analysis of IKFM ascites and tumors showed sustained shifts in macrophage populations away from the M2 and towards the anti-tumor M1 phenotype. There were also increased tumor-infiltrating CD3+/CD8+ T cells in IKFM mice, accompanied by higher levels of CXCL9, a T cell activating factor secreted by macrophages, in IKFM ascitic fluid. Conclusions In syngeneic ovarian cancer models, increased canonical NF-κB signaling in macrophages promoted anti-tumor TAM phenotypes and increased cytotoxic T cell infiltration, which was sufficient to limit tumor progression. This may present a novel translational approach for ovarian cancer treatment, with the potential to increase responses to T cell-directed therapy in future studies.

Protein–protein interactions (PPIs) provide a rich source of potential targets for drug discovery and biomedical science research. However, the identification of structural‐diverse starting points for discovery of PPI inhibitors remains a significant challenge. Activity‐directed synthesis (ADS), a function‐driven discovery approach, was harnessed in the discovery of the p53/hDM2 PPI. Over two rounds of ADS, 346 microscale reactions were performed, with prioritisation on the basis of the activity of the resulting product mixtures. Four distinct and novel series of PPI inhibitors were discovered that, through biophysical characterisation, were shown to have promising ligand efficiencies. It was thus shown that ADS can facilitate ligand discovery for a target that does not have a defined small‐molecule binding site, and can provide distinctive starting points for the discovery of PPI inhibitors., Activity‐directed synthesis (ADS) was harnessed in the discovery of the p53/hDM2 protein–protein interaction. Four novel series of PPI inhibitors were discovered that had promising ligand efficiencies. It was demonstrated that ADS can enable scaffold hopping, and facilitate lead discovery for targets lacking defined small‐molecule binding sites.

Objective. High-grade serous ovarian cancer (HGSOC) is the most common and lethal histological subtype of epithelial ovarian cancer. HGSOC with cyclin E1 gene (CCNE1) amplification and bromodomain and extraterminal 4 (BRD4) amplification have been associated with poor outcomes. Our objective was to evaluate clinical outcomes of HGSOC with co-amplification of CCNE1 and BRD4 and high protein expression of cyclin E and BRD4. Methods. Copy number amplification data were extracted from The Cancer Genome Atlas (TCGA) for 579 HGSOC. Reverse phase protein array (RPPA) TCGA data were used to determine cyclin E and BRD4 protein expression in 482 HGSOC. Cyclin E and BRD4 protein expression by immunohistochemistry (IHC) was evaluated in a tissue microarray (TMA) of 110 HGSOC. Measured clinical outcomes were survival and platinum sensitivity. Results. Of 30% of HGSOC with amplifications in CCNE1 or BRD4, 8% have both CCNE1 and BRD4 amplification. Protein expression of cyclin E and BRD4 are positively correlated, both by RPPA (r = 0.23; p < 0.001) and by IHC (r = 0.21; p = 0.025). Patients with CCNE1 and BRD4 co-amplified HGSOC have worse overall survival than patients without amplifications, 39.94 vs 48.06 months (p = 0.029). High protein expression of cyclin E, but not BRD4, was associated with poor overall survival (HR 1.62, 1.04–2.53, p = 0.033) and platinum resistance (p = 0.016). Conclusion. HGSOC with CCNE1 and BRD4 co-amplification are associated with poor overall survival. Further studies are warranted to determine the use of protein expression by IHC as a surrogate marker for CCNE1 and BRD4 co-amplified HGSOC.