Target 2035 - update on the quest for a probe for every protein.

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome.

Discovery of a Potent and Selective Covalent p300/CBP Inhibitor.

Aberrant gene activation driven by the histone acetyltransferases p300 and CREB binding protein (CBP) has been linked to several diseases, including cancers. Because of this, many efforts have been aimed toward the targeting of the closely related paralogues, p300 and CBP, but these endeavors have been exclusively directed toward noncovalent inhibitors. X-ray crystallography of A-485 revealed that both p300 and CBP possess a cysteine (C1450) near the active site, thus rendering covalent inhibition an attractive chemical approach. Herein we report the development of compound 2, an acrylamide-based inhibitor of p300/CBP that forms a covalent adduct with C1450. We demonstrated using mass spectrometry that compound 2 selectively targets C1450, and we also validated covalent binding using kinetics experiments and cellular washout studies. The discovery of covalent inhibitor 2 gives us a unique tool for the study of p300/CBP biology.

Expanding the Repertoire for "Large Small Molecules": Prodrug ABBV-167 Efficiently Converts to Venetoclax with Reduced Food Effect in Healthy Volunteers.

Since gaining approval for the treatment of chronic lymphocytic leukemia (CLL), the BCL-2 inhibitor venetoclax has transformed the treatment of this and other blood-related cancers. Reflecting the large and hydrophobic BH3-binding groove within BCL-2, venetoclax has significantly higher molecular weight and lipophilicity than most orally administered drugs, along with negligible water solubility. Although a technology-enabled formulation successfully achieves oral absorption in humans, venetoclax tablets have limited drug loading and therefore can present a substantial pill burden for patients in high-dose indications. We therefore generated a phosphate prodrug (3, ABBV-167) that confers significantly increased water solubility to venetoclax and, upon oral administration to healthy volunteers either as a solution or high drug-load immediate release tablet, extensively converts to the parent drug. Additionally, ABBV-167 demonstrated a lower food effect with respect to venetoclax tablets. These data indicate that beyond-rule-of-5 molecules can be successfully delivered to humans via a solubility-enhancing prodrug moiety to afford robust exposures of the parent drug following oral dosing.

Long-Term Outcomes of Patients With Early Stage Nonbulky Hodgkin Lymphoma Treated With Combined Modality Therapy in the Stanford V Trials (the G4 and G5 Studies).

PURPOSE: Combined modality therapy (CMT) is standard therapy for early-stage Hodgkin lymphoma (ESHL). We previously reported excellent outcomes with the abbreviated Stanford V regimen. Herein we report updated results with median follow-up >10 years on survival, therapy-related late effects, and impact of disease risk factors on patient outcomes. METHODS AND MATERIALS: The G4 and G5 studies enrolled patients with stage I-IIA nonbulky ESHL. Patients received 8 weeks of Stanford V chemotherapy followed by 30 Gy modified involved-field radiation therapy (mIFRT) (G4) or Stanford V-C + 20 Gy mIFRT (G5). Patients were categorized as favorable or unfavorable risk per German Hodgkin Study Group (GHSG) criteria and outcomes between groups compared. RESULTS: A total of 129 patients were enrolled (68 favorable and 61 unfavorable risk). In the G4 study (n = 87), at median follow-up of 19.7 years, 5-, 10-, and 15-year progression-free survival (PFS) and overall survival (OS) were 95.4%/97.7%, 91.8%/96.5%, and 91.8%/95.3%, respectively. In the G5 study (n = 42), at median follow-up of 13.5 years, the 5-, 10-, and 15-year PFS and OS were 92.9%/100%, 92.9%/100%, and 88.4%/91.9%, respectively. PFS (P = .86) and OS (P = .86) were not significantly different between studies. There were also no significant differences between studies in patients with favorable or unfavorable risk for PFS (F: P = .53; U: P = .96), OS (F: P = .99; U: P = .78), secondary malignancies (F: P = .74; U: P = 1.0), and cardiovascular complications (F: no cases; U: P = 1.0). CONCLUSIONS: The G4 and G5 studies achieve high rates of durable remission; 20 versus 30 Gy mIFRT and cyclophosphamide substituted for mechlorethamine did not compromise nodal control, PFS, or OS in both favorable and unfavorable risk disease. These results support the efficacy of CMT in early-stage disease and lower-dose radiation therapy in patients with favorable and nonbulky unfavorable ESHL.

Long-term outcomes of patients with unfavorable stage I-II classic Hodgkin lymphoma treated with Stanford V chemotherapy and limited field irradiation.

Management of stage I-II unfavorable risk Hodgkin lymphoma (HL) strives to reduce toxicity while maintaining tumor control. Compared to ABVD or BEACOPP, Stanford V chemotherapy contains less doxorubicin and bleomycin. We report long-term outcomes of patients with stage I-II classic HL with European risk factors treated with Stanford V combined modality therapy (CMT). From our institutional cancer registry, we identified 168 patients with ≥1 European risk factor treated with 8-12 weeks of Stanford V CMT and consolidative radiotherapy between 1990 and 2016. Outcomes were analyzed after classification by EORTC and GHSG unfavorable criteria. With median follow-up of 8.4 years, 10-year overall survival and progression-free survival for the entire cohort were 95% and 88%, respectively. Thirteen of 18 relapses were salvaged successfully. There were no cases of MDS or AML after primary therapy. Long-term outcomes of stage I-II unfavorable risk HL treated with Stanford V CMT are comparable to ABVD or BEACOPP regimens.

Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer.

Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clinical activity in monotherapy cancer trials. A reduced number of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi1-5. Given that similar haematological and gastrointestinal defects were observed after genetic silencing of Brd4 in mice6, the platelet and gastrointestinal toxicities may represent on-target activities associated with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions7-9 and different cellular phenotypes after pharmacological inhibition of one or both bromodomains have been reported10,11, suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compounds that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment10-13. Here we carried out a medicinal chemistry campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukaemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-07514. Analyses of RNA expression and chromatin immunoprecipitation followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-containing super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy.

Prognostic factors and patterns of failure in advanced stage Hodgkin lymphoma treated with combined modality therapy.

BACKGROUND AND PURPOSE: The role of irradiation to non-bulky and bulky sites of disease in advanced stage Hodgkin lymphoma is controversial. We aimed to review the long-term outcomes of patients treated with combined modality therapy to clarify the role of consolidative radiotherapy. MATERIALS AND METHODS: Patients with stage III or IV Hodgkin lymphoma treated with Stanford V chemotherapy and consolidative radiotherapy to initial sites of disease ≥5 cm were analyzed retrospectively to determine patient outcomes, patterns of failure, and factors associated with treatment failure. RESULTS: A total of 170 patients were analyzed. Overall survival was 91.2%, freedom from progression was 80.6%, and progression-free survival was 78.9% at 10 years. 5 patients (2.9%) had refractory disease and 27 patients (15.9%) relapsed after treatment. Only an International Prognostic Score (IPS) greater than 2 predicted disease progression. 19 out of 27 relapses occurred exclusively outside of the radiation treatment field, and 17 out of 27 relapses occurred exclusively at original sites of disease. However, only 11 of 170 patients (6.5%) relapsed exclusively at original, non-bulky sites of disease not treated with radiation therapy. The cumulative incidence of local failure at 10 years was 4.6% for unirradiated sites and 2.6% for irradiated sites. CONCLUSION: Patients with advanced stage Hodgkin lymphoma treated with combined modality therapy including consolidative radiotherapy to bulky disease sites had excellent long-term outcomes. Given the low frequency of isolated failures at initial sites, our results suggest that selective radiation therapy to sites at high risk of relapse may be feasible.

Author Correction: Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

In the originally published version of this Letter, the authors Arthur F. Kluge, Michael A. Patane and Ce Wang were inadvertently omitted from the author list. Their affiliations are: I-to-D, Inc., PO Box 6177, Lincoln, Massachusetts 01773, USA (A.F.K.); Mitobridge, Inc. 1030 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (M.A.P.); and China Novartis Institutes for BioMedical Research, No. 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong District, Shanghai 201203, China (C.W.). These authors contributed to the interpretation of results and design of compounds. In addition, author 'Edward A. Kesicki' was misspelled as 'Ed Kesicki'. These errors have been corrected online.

Donated chemical probes for open science.

Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (https://openscienceprobes.sgc-frankfurt.de/). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project.

Found in Translation: How Preclinical Research Is Guiding the Clinical Development of the BCL2-Selective Inhibitor Venetoclax.

Since the discovery of apoptosis as a form of programmed cell death, targeting the apoptosis pathway to induce cancer cell death has been a high-priority goal for cancer therapy. After decades of effort, drug-discovery scientists have succeeded in generating small-molecule inhibitors of antiapoptotic BCL2 family proteins. Innovative medicinal chemistry and structure-based drug design, coupled with a strong fundamental understanding of BCL2 biology, were essential to the development of BH3 mimetics such as the BCL2-selective inhibitor venetoclax. We review a number of preclinical studies that have deepened our understanding of BCL2 biology and facilitated the clinical development of venetoclax.Significance: Basic research into the pathways governing programmed cell death have paved the way for the discovery of apoptosis-inducing agents such as venetoclax, a BCL2-selective inhibitor that was recently approved by the FDA and the European Medicines Agency. Preclinical studies aimed at identifying BCL2-dependent tumor types have translated well into the clinic thus far and will likely continue to inform the clinical development of venetoclax and other BCL2 family inhibitors. Cancer Discov; 7(12); 1376-93. ©2017 AACR.

Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.

Discovery and Characterization of Novel Nonsubstrate and Substrate NAMPT Inhibitors.

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell death when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236-45. ©2017 AACR.

Preclinical Characterization of BET Family Bromodomain Inhibitor ABBV-075 Suggests Combination Therapeutic Strategies.

ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G1 cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. In vivo combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of in vitro synergy between ABBV-075 and these agents. The in vitro/in vivo activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. Cancer Res; 77(11); 2976-89. ©2017 AACR.

Breast Imaging in Women Previously Irradiated for Hodgkin Lymphoma.

BACKGROUND: Women treated with mantle irradiation for Hodgkin lymphoma (HL) are at an increased risk of developing breast cancer (BC). Current guidelines recommend screening breast magnetic resonance imaging (MRI) as an adjunct to mammography (M) in these patients. There are limited data, however, as to the impact of breast MRI on cancer detection rates. The aim of the current study is to evaluate the use of breast MRI in survivors of HL treated and followed at a single institution. MATERIALS AND METHODS: We retrospectively reviewed 980 female patients treated with mantle irradiation for HL between 1961 and 2008. Records were reviewed to determine age at radiotherapy treatment, radiotherapy dose, breast imaging (including M and breast MRI), biopsy results if applicable, and incidence of BC. RESULTS: A total of 118 patients had breast imaging performed at our institution. Median age at HL diagnosis was 28 years (range, 10 to 69 y). Median radiotherapy dose was 36 Gy (range, 20 to 45 Gy). Seventy-nine patients (67%) underwent M screening only, 1 (1%) breast MRI only, and 38 (32%) both M and breast MRI. Of these 38, 19 (50%) underwent 54 screening MRI studies (range per patient=1 to 8), 13 (34%) underwent preoperative MRI for workup of BC, and 6 (16%) initiated screening MRI of the contralateral breast only after diagnosed with BC. Fifty-nine biopsies were performed: 47 were prompted by suspicious M findings only, 10 by palpable findings on physical examination (PE), and 2 by suspicious breast MRI findings. Of the 47 biopsies prompted by M, 24 revealed malignant disease, whereas 23 proved to be benign. All 10 biopsies performed by palpation were malignant. Both biopsies prompted by MRI findings were benign. With M, there were 34 true-positive findings in 32 patients, 23 false-positive findings, and 1 false-negative finding. With screening MRI, there were 2 false-positive findings, 1 false-negative finding, and no true-positive findings. CONCLUSIONS: The role of screening breast MRI in women previously irradiated for HL is evolving. Further education of patients and physicians is important to increase awareness of more sensitive BC screening modalities in this high-risk population. Future studies are necessary to determine the appropriate integration of screening breast MRI into the ongoing follow-up of these women.

Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.

Structure-guided design of a series of MCL-1 inhibitors with high affinity and selectivity.

Myeloid cell leukemia 1 (MCL-1) is a BCL-2 family protein that has been implicated in the progression and survival of multiple tumor types. Herein we report a series of MCL-1 inhibitors that emanated from a high throughput screening (HTS) hit and progressed via iterative cycles of structure-guided design. Advanced compounds from this series exhibited subnanomolar affinity for MCL-1 and excellent selectivity over other BCL-2 family proteins as well as multiple kinases and GPCRs. In a MCL-1 dependent human tumor cell line, administration of compound 30b rapidly induced caspase activation with associated loss in cell viability. The small molecules described herein thus comprise effective tools for studying MCL-1 biology.