Abstract The average timeline from project initiation to preclinical candidate delivery in oncology drug discovery is approximately four years. To compress these timelines, direct-to-biology strategies are increasingly employed to expedite early decision-making and compound progression. However, conventional approaches often remain limited to biophysical off-rate assessments of crude reaction mixtures (CRMs), restricting their utility. To overcome these constraints, we have expanded this paradigm through integrated methodologies, including Direct-to-Crystallography (D2C), to validate hits and elucidate structure-activity relationships (SAR), enabling pre-candidate delivery in as little as 18 months. Harnessing our Hit Synergy platform which combines complementary screening technologies, we rapidly generated novel, high quality and structurally diverse hits for a challenging and clinically relevant oncology target. Early deployment of D2C, using existing HTS liquid stocks, delivered ligand-bound structures without delays associated with hit re-synthesis or re-acquisition. These structures confirmed target engagement and revealed novel binding interactions. Orthogonal hit validation employed direct-to-biology approaches such as off-rate screening, alongside tailored biochemical and cellular assays on purified samples. Integration of structural insights and biological data accelerated hit expansion via high-throughput chemistry (HTC) and complementary direct-to-biology assays validated for both pure compounds and CRMs. Robust D2C determinations from CRMs yielding as little as 15% product further streamlined SAR generation and hypothesis testing, influencing medicinal chemistry design and decision making and compressing the design-make-test cycle. Generative AI-driven idea generation and direct-to-physicochemical assays further supported optimization for developability. Together, this fully integrated, multi-modal workflow enabled rapid, holistic decision-making at critical project stages, delivering pre-candidate quality derivatives in a significantly truncated timeframe. By embedding direct-to-discovery methodologies throughout the pipeline, we successfully accelerated progression from hit identification to candidate selection, advancing novel therapeutics toward the clinic with unprecedented efficiency. Citation Format: Allan Michael Jordan, Daniel Clare, Kam Chohan, Rick Davies, Jonathan Finlayson, Euan Fordyce, Clare Hammond, Philip MacFaul, Christopher Pearce, Raquel Faba Rodriguez, Alexandra Stowell, Denise Swift, Megan Thompson, Graeme Walker, Terence Wu. DiscoveryDirect: Extending direct-to-biology paradigms to accelerate oncology drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6428.

Differentially expressed genes with AZD5153 treatment

Supplementary Table from AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies

Cell line authentication information

Protein modulation by AZD5153 in hematological cell lines

Legends for Supplementary Figures 1 - 7.

Supplementary Figure 1 contains a Western blot showing the amount of estrogen receptor remaining after treatment of MCF-7 cells with AZD9496, fulvestrant and estradiol alongside GAPDH protein levels as an internal gel loading control; Supplementary Figure 2 shows the rate of decreased detection by mass spectrometry of an estrogen receptor heavy isotype labelled peptide over time after treatment of cells with AZD9496, fulvestrant and tamoxifen and the calculated half life; Supplementary Figure 3 shows the degradation of estrogen receptor α by AZD9496, fulvestrant and estradiol in the presence of +/- 10 μM proteosomal inhibitor MG132; Supplementary Figure 4 contains a Western blot showing the re-emergence over 48 hours of estrogen receptor α following removal of AZD9496, fulvestrant and estradiol compounds from MCF-7 cells; Supplementary Figure 5 shows a bar chart of the measured estradiol levels in the blood of mice over 42 days after implantation of the mice with estrogen pellets to support growth of the MCF-7 cells as a tumour in mice; Supplementary Figure 6 shows the effects of AZD9496, fulvestrant and tamoxifen on mRNA levels of estrogen receptor regulated genes in vitro and in vivo; Supplementary Figure 7 shows the levels of estrogen receptor α protein remaining in tumours from the HCC1428 LTED model after treatment with AZD9496 and fulvestrant.

PDF - 464KB, Supplementary Figure 1: Inhibition of AR transcripts with AZD3514 treated LNCaPs in steroid free media and the timecourse of AZD3514 effects in the presence and absence of DHT. Supplementary Figure 2: Effect of AZD3514 on growth of AR negative cells. Supplementary Figure 3: AR and PSA expression in LAPC4 cells treated with AZD3514. Supplementary Figure 4: AR degradation and synthesis rates of a C-terminal AR peptide, as measured by Mass Spectrometry. Supplementary Figure 5: Comparison of ARD1 & AZD3514 effects on seminal vesicle weight in rats, and pharmacokinetic effects in mice and rats Supplementary Figure 6: Effect of ARD1 on growth and AR in the HID28 model. Supplementary Figure 7: Dose response effects of AZD3514 on AR nuclear translocation and foci formation.

Abstract Introduction: Radiotherapy is a main treatment modality for multiple cancer types, with approximately half of all cancer patients receiving radiotherapy as part of their treatment regimen. In addition to direct cytotoxic effects on cancer cells, it is now appreciated that radiosensitization using specific drugs inhibiting DNA damage response (DDR) pathways can enhance the sensitivity of tissues or cells to radiation therapy. Establishing assays to screen for potential radiosensitizing compounds is therefore valuable to inform drug discovery research. Methods: The MDA-MB-436 human breast cancer cell line was selected for assay optimization. DDR inhibitors of DNA-dependent protein kinase (DNA-PK) (AZD7648) and Poly-(ADP-Ribose)-Polymerase (PARP) (niraparib) were used as tool radiosensitizing compounds, as these proteins play key roles in the repair of radiation-induced DNA double strand breaks. Effects on cell viability, cell cycle progression and DDR biomarkers were determined under a variety of irradiation (IR) and compound conditions. In parallel, in vivo validation experiments were performed. Results and Conclusions: Both AZD7648 and niraparib radiosensitize MDA-MB-436 cells to IR treatment. A dose of 2 Gray (Gy) reduced the observed anti-proliferative compound IC50 compared to IR treatment or compound treatment alone. Furthermore, combined treatment of 2 Gy IR with either DDR inhibitor induces S-phase cell cycle arrest and increases cell death. Analysis of DDR biomarkers confirms their induction by radiotherapy in combination with DDR inhibitors. Similar changes to DDR biomarker expression were also observed in the in vivo xenograft mouse model. In summary, in vitro and in vivo assays have been established to investigate radiosensitization through the combination of IR and DDR inhibitors. Furthermore, these assays can identify novel compounds acting via a synergistic mechanism. Citation Format: Charlotte Bell, Stewart Brown, Amy Cantrell, Paul Farrington, Miguel Garcia, Ben Hodgson, Yin Xin Ho, Allan Jordan, Theoni Katopodi, Jane Kendrew, Wen Hao Neo, Stuart Thomson, Graeme E Walker, Emily Wright. Establishing assays to investigate combinations of fractioned radiotherapy with DNA damage response agents in vitro and in vivo to enable investigation of radiosensitization and improved anti-tumour responses [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A151.

Supplementary Table 1 contains crystallographic data collection and refinements statistics for AZD9496 binding to the human estrogen receptor α ligand binding domain, Supplementary Table 2 shows the rate constants for association, dissociation and equilibrium dissociation constant for AZD9496 binding to the human estrogen receptor α ligand binding domain, Supplementary Table 3 displays IC50 values for AZD9496 binding to androgen, glucocorticoid, progesterone and estrogen receptor β ligand binding domains, Supplementary Table 4 displays IC50s values for cell growth inhibition of a panel of estrogen receptor positive and negative cell lines using AZD9496.

PDF - 56KB, Supplementary methods detailing Relative peptide quantification by selected reaction monitoring (SRM)using mass spectrometry

Supplementary Figure 1. AZD5153 shows reduced BRD4 binding activity when BD2 function is abolished; Supplementary Figure 2. AZD5153 modulates MYC protein levels across hematologic cancer cell lines; Supplementary Figure 3. Gene Ontology (GO) analysis of the down-regulated transcripts by AZD5153 across all cell line types; Supplementary Figure 4. IC50 and percent maximal cell kill by AZD5153 and I-BET762 in five hematologic cell lines; Supplementary Figure 5. Tumor growth inhibition across six hematological xenograft Models

PDF - 329KB, Supplementary Table 1: TSQ Vantage mass spectrometry instrument settings. Supplementary Table 2: 4000 QTRAP mass spectrometry instrument settings. Supplementary Table 3: The half-life of AR for the first 6 hours following treatment calculated for both the N-terminal (i) and C-terminal (ii) AR peptides assuming exponential decay. Supplementary Table 4: Summary table of pharmacokinetics of ARD1 and AZD3514 in the mouse.

Structure-based drug discovery (SBDD) largely relies on structural information from X-ray crystallography because traditional NMR structure calculation methods are too time consuming to be aligned with typical drug discovery timelines. The recently developed NMR molecular replacement (NMR2) method dramatically reduces the time needed to generate ligand-protein complex structures using published structures (apo or holo) of the target protein and treating all observed NOEs as ambiguous restraints, bypassing the laborious process of obtaining sequence-specific resonance assignments for the protein target. We apply this method to two therapeutic targets, the bromodomain of TRIM24 and the second bromodomain of BRD4. We show that the NMR2 methodology can guide SBDD by rationalizing the observed SAR. We also demonstrate that new types of restraints and selective methyl labeling have the potential to dramatically reduce "time to structure" and extend the method to targets beyond the reach of traditional NMR structure elucidation.

HER2+ BC is associated with a high incidence (up to 50%) of brain metastasis (BM) despite advances in treatment (Freedman et al. J Clin Oncol. 2019). Although several agents have been studied in patients (pts) with HER2+ BC with BM, an unmet medical need remains due to the poor prognosis in this pt population. In DESTINY-Breast01, T-DXd demonstrated efficacy in the overall population and preliminary efficacy in a pt subgroup with stable BM, with a confirmed objective response rate (ORR) of 61.4% and an extracranial confirmed ORR by independent central review (ICR) of 58.3%, respectively, median progression-free survival (PFS) of 19.4 and 18.1 mo, respectively, and median duration of response (DOR) of 20.8 and 16.9 mo, respectively (Modi S et al. Cancer Res. 2021. Abst PD3-06; Jerusalem et al. Ann Oncol. 2020. Abst 138O). Here we describe a trial evaluating T-DXd in pts ± BM with previously treated advanced/metastatic HER2+ BC. DESTINY-Breast12 (NCT04739761) is an open-label, multicenter, international (86 sites in the US, Europe, Australia, and Japan), phase 3b/4 study assessing the efficacy and safety of T-DXd 5.4 mg/kg q3w in pts with HER2+ BC ± BM. Pts will be enrolled in 1 of 2 cohorts (250 pts each): cohort 1 (−BM at baseline) and cohort 2 (+BM at baseline). Pts must have previously treated advanced/metastatic HER2+ BC that has progressed with ≥1 prior anti-HER2–based regimen and received ≤2 lines of therapy in the metastatic setting (excludes pts with prior tucatinib). Pts with BM must have untreated BM not needing immediate local therapy or previously treated stable or progressing BM. Primary endpoints are ORR in cohort 1 and PFS in cohort 2 (both by RECIST version 1.1 per ICR). Secondary endpoints in both cohorts are OS, DOR, time to progression, duration of subsequent therapy, PFS2, safety, and changes in symptoms, functioning, and QOL. Incidence of new symptomatic CNS metastasis (CNSM) is a secondary endpoint in cohort 1, and ORR and CNS ORR by RECIST 1.1 per ICR, CNS PFS and DOR, and time to new CNSM are secondary endpoints in cohort 2. NCT04739761. Medical editorial assistance was provided by ArticulateScience LLC, and funded by AstraZeneca Pharmaceuticals LP. AstraZeneca. AstraZeneca.

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.

Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC50 of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.

Abstract The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563–74. ©2016 AACR.

In recent years newer mutation-specific and targeted therapies have become available for the management of ovarian cancer (OC). However, there are limited data documenting recent treatment patterns among women with OC. A retrospective observational study was conducted using the Humedica EHR database (2007-2014). Women diagnosed with OC (ICD-9-CM code 183.0x), enrolled in an integrated delivery network and receiving chemotherapy or cancer directed surgery were included in the study. Patients were followed from index OC diagnosis date until end of database, death date or database activity end date, whichever occurred earlier. Study measures assessed included patient demographic and clinical characteristics and post-diagnosis treatment patterns including treatments received (surgery, chemotherapy, targeted and radiation therapy), treatment sequence, line of therapy. All analyses were descriptive in nature. The study cohort included 2,158 patients with an average age of 62 (SD: 13) years and average post-diagnosis follow-up of 29 months in the database. Post diagnosis OC-related treatments included: chemotherapy (67%), surgery (65%), radiation therapy (10%) and targeted therapy (bevacizumab 9% [approved in the US for platinum-resistant recurrent OC in combination with paclitaxel, pegylated liposomal doxorubicin or topotecan]). Among patients initiating first-line therapy, carboplatin alone or in combination with other agents accounted for ~63% of first-line regimens. Commonly observed first-line non-platinum based therapies included paclitaxel (5%), doxorubicin (3%) and gemcitabine (2%). Among first-line therapy initiators 42% had evidence of second-line therapy in the database. Similarly, among patients with second-line therapy, 56% had evidence for a third-line therapy in the database with doxorubicin (20%) and carboplatin+paclitaxel (15%) being the commonly observed third-line therapies. This study provides recent treatment patterns data among women with OC in the US. With newer therapies being developed for OC, findings from this study can serve as inputs in the development of budget impact models comparing new and existing treatments.

Abstract The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Across a number of tumor models pharmacological targeting of BRD4 bromodomains by small-molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor. AZD5153 is a candidate drug that possesses an unprecedented bivalent binding mode among reported BET inhibitors, which allows AZD5153 to ligate the tandem bromodomains in BRD4. The avidity resulted from the bivalent binding interaction translates into markedly enhanced cellular potency. Although AZD5153 demonstrates broad activity across a cancer cell line panel comprising solid and hematologic subtypes, there is enriched antitumor activity against hematologic cell lines, including acute myeloid leukemia (AML), multiple myeloma (MM), and diffuse large B-cell lymphoma (DLBCL). The activity of AZD5153 in hematologic tumors was further confirmed in five selected xenograft models of AML, MM, and DLBCL where AZD5153 treatment led to tumor stasis or regression, accompanied by concomitant modulation of BRD4 pharmacodynamic markers, such as MYC and HEXIM1. In order to characterize the transcriptional consequences elicited by AZD5153, we carried out transcriptional profiling of 11 hematologic tumor lines and identified the robust modulation of MYC, and E2F transcriptional programs. Moreover, our transcriptional data was used to identify candidate clinical PD biomarkers. The suitability and dynamic range of the top two candidate biomarkers for AZD5153 was confirmed using human whole blood from normal healthy volunteers. To identify protein biomarkers associated with sensitivity to AZD5153 treatment, we deployed reverse-phase protein array (RPPA) technology to quantitatively examine the level of 182 proteins following AZD5153 treatment. Our findings indicate that cell lines sensitive to AZD5153 uniquely exhibit a marked decrease in the level of mTOR-pathway associated proteins following AZD5153 treatment. Conversely, MYC modulation was observed in both sensitive and resistant groups. Thus, these data suggest that in hematologic malignancies, mTOR pathway downregulation may serve as an appropriate biomarker of sensitivity to BRD4 inhibitors such as AZD5153. Our study establishes AZD5153 as a novel and potent BRD4/BET inhibitor possessing a unique bivalent binding property. We have characterized the pharmacological consequences of BRD4/BET inhibition by AZD5153 via unbiased transcriptional and proteome profiling. These efforts are the first to identify mTOR modulation as a putative biomarker of sensitivity to BET bromodomain inhibition in hematologic tumors and may help to inform future clinical evaluation of AZD5153 and other BET bromodomain inhibitors. Citation Format: Huawei (Ray) Chen, Maureen Hattersley, Garrett Rhyasen, Austin Dulak, Wendy Wang, Phil Petteruti, Ian Dale, Tony Cheung, Shenghua Wen, Lilian Castriotta, Deborah Lawson, Mike Collins, Miika Ahdesmaki, Graeme Walker, Al Rabow, Jonathan Dry, Corinne Reimer, Paul Lyne, Steve Fawell, MIke Waring, Mike Zinda, Ed Clark, Ed Clark. Therapeutic activity of bivalent BRD4 inhibitor AZD5153 in hematological cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4705.