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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., (©2021 American Association for Cancer Research.)

BCL-X L , an antiapoptotic member of the BCL-2 family of proteins, drives tumor survival and maintenance and thus represents a key target for cancer treatment. Herein we report the rational design of a novel series of selective BCL-X L inhibitors exemplified by A-1293102. This molecule contains structural elements of selective BCL-X L inhibitor A-1155463 and the dual BCL-X L /BCL-2 inhibitors ABT-737 and navitoclax, while representing a distinct pharmacophore as assessed by an objective cheminformatic evaluation. A-1293102 exhibited picomolar binding affinity to BCL-X L and both efficiently and selectively killed BCL-X L -dependent tumor cells. X-ray crystallographic analysis demonstrated a key hydrogen bonding network in the P2 binding pocket of BCL-X L , while the bent-back moiety achieved efficient occupancy of the P4 pocket in a manner similar to that of navitoclax. A-1293102 represents one of the few distinct structural series of selective BCL-X L inhibitors, and thus serves as a useful tool for biological studies as well as a lead compound for further optimization., Competing Interests: The authors declare the following competing financial interest(s): AbbVie participated in interpretation of data and review and approval of publication. Z-F.T., X.W., J.C., J.P.I., S.J., R.A.J., P.J.K., C.S., B.D.W., L.Z., H.Z., S.W.E., D.C.P., A.S.J., J.D.L., and A.J.S. are employees of AbbVie; C.P. was an employee of AbbVie at the time of the study., (© 2021 American Chemical Society.)

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-X L inhibitor that selectively and potently induces apoptosis in BCL-X L -dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-X L inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp 3 -rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-X L . A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program., Competing Interests: The authors declare the following competing financial interest(s): AbbVie and Genentech participated in interpretation of data and review and approval of publication. L.W., G.A.D., A.S.J., Z-F.T., T. M. H., R.R.F., X.S., M.B., A.R.K., X.W., M.D.W., N.D.C., S.S., D.C.P., R.A.J., P.N., M.L.S., S.K.T., Y.X., J.X., H.Z., P.N.L., M.J.M., E.R.B., W.G., P.K., S.W.E., J.D.L., and A.J.S. are employees of AbbVie, Inc. C.H.P. was an employee of AbbVie, Inc at the time of the study. E.C. and W.J.F. are employees of Genentech, Inc. J.A.F., D.D., and D.S. were employees of Genentech, Inc. at the time of the study.

The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examination in clinical studies, particularly in oncology settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metabolism and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a critical asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Additional structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clinical candidate 63 (ABBV-075/mivebresib), which demonstrates excellent potency in biochemical and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.

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 G 1 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 ., (©2017 American Association for Cancer Research.)

An NMR fragment screen for binders to the bromodomains of BRD4 identified 2-methyl-3-ketopyrroles 1 and 2. Elaboration of these fragments guided by structure-based design provided lead molecules with significant activity in a mouse tumor model. Further modifications to the methylpyrrole core provided compounds with improved properties and enhanced activity in a mouse model of multiple myeloma., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, K i = 160 μM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.

Hyperexpression of antiapoptotic BCL-2 family proteins allows cells to survive despite the receipt of signals that would ordinarily induce their deletion, a facet frequently exploited by tumors. Tumors addicted to the BCL-2 family proteins for survival are now being targeted therapeutically. For example, navitoclax, a BCL-2/BCL-XL/BCL-W inhibitor, is currently in phase I/II clinical trials in numerous malignancies. However, the related family member, MCL-1, limits the efficacy of navitoclax and other chemotherapeutic agents. In the present study, we identify breast cancer cell lines that depend upon MCL-1 for survival and subsequently determine the mechanism of apoptosis mediated by the MCL-1 selective inhibitor A-1210477. We demonstrate that apoptosis resulting from a loss in MCL-1 function requires expression of the proapoptotic protein BAK. However, expression of BCL-XL can limit apoptosis resulting from loss in MCL-1 function through sequestration of free BIM. Finally, we demonstrate substantial synergy between navitoclax and MCL-1 siRNA, the direct MCL-1 inhibitor A-1210477, or the indirect MCL-1 inhibitor flavopiridol, highlighting the therapeutic potential for inhibiting BCL-XL and MCL-1 in breast cancer., (©2015 American Association for Cancer Research.)

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., (Copyright © 2015, American Association for the Advancement of Science.)

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.

The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.

A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.

Because of the promise of BCL-2 antagonists in combating chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), interest in additional selective antagonists of antiapoptotic proteins has grown. Beginning with a series of selective, potent BCL-XL antagonists containing an undesirable hydrazone functionality, in silico design and X-ray crystallography were utilized to develop alternative scaffolds that retained the selectivity and potency of the starting compounds.

Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.

The ability of a cancer cell to avoid apoptosis is crucial to tumorigenesis and can also contribute to chemoresistance. The Bcl-2 family of prosurvival proteins (Bcl-2, Bcl-X(L), Bcl-w, Mcl-1, and A1) plays a key role in these processes. We previously reported the discovery of ABT-263 (navitoclax), a potent small-molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w. While navitoclax exhibits single-agent activity in tumors dependent on Bcl-2 or Bcl-X(L) for survival, the expression of Mcl-1 has been shown to confer resistance to navitoclax, most notably in solid tumors. Thus, therapeutic agents that can downregulate or neutralize Mcl-1 are predicted to synergize potently with navitoclax. Here, we report the activity of navitoclax in combination with 19 clinically relevant agents across a panel of 46 human solid tumor cell lines. Navitoclax broadly enhanced the activity of multiple therapeutic agents in vitro and enhanced efficacy of both docetaxel and erlotinib in xenograft models. The ability of navitoclax to synergize with docetaxel or erlotinib corresponded to an altered sensitivity of the mitochondria toward navitoclax, which was associated with the downmodulation of Mcl-1 and/or upregulation of Bim. These data provide a rationale to interrogate these combinations clinically.

Non-small-cell lung cancer (NSCLC) is the most deadly type of cancer in the United States and worldwide. Although new therapy is available, the survival rate of NSCLC patients remains low. One hallmark of cancer cells is defects in the apoptotic cell death program. In this study, we investigate the role of B-cell lymphoma 2 (Bcl-2) family members Bcl-2, Bcl-x(L) and Mcl-1, known to regulate cell survival and death, in a panel of fourteen NSCLC cell lines. NSCLC cell lines express high levels of Mcl-1 and Bcl-x(L), but not Bcl-2. Silencing the expression of Mcl-1 with small interfering RNA (siRNA) oligonucleotides potently killed a subgroup of NSCLC cell lines. In contrast, Bcl-x(L) siRNA had no effect in these lines unless Mcl-1 siRNA was also introduced. Interestingly, high MCL1 to BCL-xl messenger RNA determines whether the cells depend on Mcl-1 for survival. We further investigated the role of Mcl-1 in NSCLC cells using a Mcl-1-dependent cell line, H23. The expression of a complementary DNA containing only the coding region of MCL1 rescued H23 cells from the toxicity of a 3' untranslated region (UTR) targeting Mcl-1 siRNA but not a siRNA targeting the coding region of MCL1. Furthermore, we show that Mcl-1 sequesters the BH3-only protein Noxa and Bim and the apoptotic effector Bak. Not surprisingly, Noxa, Bim, or Bak knockdown partially rescued H23 cells from toxicity mediated by Mcl-1 siRNA to different degrees. Collectively, our results indicate that targeting Mcl-1 may improve therapy for a subset of NSCLC patients.

We describe the development of a novel series of N-aryl-benzimidazolone HSP90 inhibitors (9) targeting the N-terminal ATP-ase site. SAR development was influenced by structure-based design based around X-ray structures of ligand bound HSP90 complexes. Lead compounds exhibited high binding affinities, ATP-ase inhibition and cellular client protein degradation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Proteins of the BCL-2 family regulate clonal selection and survival of lymphocytes, and are frequently overexpressed in lymphomas. Navitoclax is a targeted high-affinity small molecule that inhibits the anti-apoptotic activity of BCL-2 and BCL-XL. We aimed to assess the safety and antitumour activity of navitoclax in patients with lymphoid tumours, and establish the drug's pharmacokinetic and pharmacodynamic profiles., Methods: In this phase 1 dose-escalation study, patients (aged ≥18 years) with relapsed or refractory lymphoid malignancies were enrolled and treated at seven sites in the USA between November, 2006, and November, 2009. A modified Fibonacci 3+3 design was used to assign patients to receive oral navitoclax once daily by one of two dosing schedules: intermittently for the first 14 days of a 21-day cycle (14/21) at doses of 10, 20, 40, 80, 110, 160, 225, 315, or 440 mg/day; or continuously for 21 days of a 21-day cycle (21/21) at doses of 200, 275, 325, or 425 mg/day. Study endpoints were safety, maximum tolerated dose, pharmacokinetic profile, pharmacodynamic effects on platelets and T cells, and antitumour activity. This trial is registered with ClinicalTrials.gov, number NCT00406809., Findings: 55 patients were enrolled (median age 59 years, IQR 51-67), 38 to receive the 14/21 dosing schedule, and 17 to receive the 21/21 dosing schedule. Common toxic effects included grade 1 or 2 anaemia (41 patients), infection (39), diarrhoea (31), nausea (29), and fatigue (21); and grade 3 or 4 thrombocytopenia (29), lymphocytopenia (18), and neutropenia (18). On the intermittent 14/21 schedule, dose-limiting toxic effects were hospital admissions for bronchitis (one) and pleural effusion (one), grade 3 increase in aminotransferases (one), grade 4 thrombocytopenia (one), and grade 3 cardiac arrhythmia (one). To reduce platelet nadir associated with intermittent 14/21 dosing, we assessed a 150 mg/day lead-in dose followed by a continuous 21/21 dosing schedule. On the 21/21 dosing schedule, two patients did not complete the first cycle and were excluded from assessment of dose-limiting toxic effects; dose-limiting toxic effects were grade 4 thrombocytopenia (one), grade 3 increase in aminotransferases (one), and grade 3 gastrointestinal bleeding (one). Navitoclax showed a pharmacodynamic effect on circulating platelets and T cells. Clinical responses occurred across the range of doses and in several tumour types. Ten of 46 patients with assessable disease had a partial response, and these responders had median progression-free survival of 455 days (IQR 40-218)., Interpretation: Navitoclax has a novel mechanism of peripheral thrombocytopenia and T-cell lymphopenia, attributable to high-affinity inhibition of BCL-XL and BCL-2, respectively. On the basis of these findings, a 150 mg 7-day lead-in dose followed by a 325 mg dose administered on a continuous 21/21 dosing schedule was selected for phase 2 study., Funding: Abbott Laboratories, Genentech, and National Cancer Institute, National Institutes of Health., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The Bcl-2 family of proteins plays a major role in the regulation of apoptosis, or programmed cell death. Overexpression of the anti-apoptotic members of this family (Bcl-2, Bcl-x(L), and Mcl-1) can render cancer cells resistant to chemotherapeutic agents and therefore these proteins are important targets for the development of new anti-cancer agents. Here we describe the discovery of a potent, highly selective, Bcl-2 inhibitor using SAR by NMR and structure-based drug design which could serve as a starting point for the development of a Bcl-2 selective anti-cancer agent. Such an agent would potentially overcome the Bcl-x(L) mediated thrombocytopenia observed with ABT-263., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Purpose: This study was designed to test the ability of the Bcl-2 family inhibitor ABT-263 to potentiate commonly used chemotherapeutic agents and regimens in hematologic tumor models., Methods: Models of B-cell lymphoma and multiple myeloma were tested in vitro and in vivo with ABT-263 in combination with standard chemotherapeutic regimens, including VAP, CHOP and R-CHOP, as well as single cytotoxic agents including etoposide, rituximab, bortezomib and cyclophosphamide. Alterations in Bcl-2 family member expression patterns were analyzed to define mechanisms of potentiation., Results: ABT-263 was additive with etoposide, vincristine and VAP in vitro in the diffuse large B-cell lymphoma line (DLBCL) DoHH-2, while rituximab potentiated its activity in SuDHL-4. Bortezomib strongly synergized with ABT-263 in the mantle cell lymphoma line Granta 519. Treatment of DoHH-2 with etoposide was associated with an increase in Puma expression, while bortezomib upregulated Noxa expression in Granta 519. Combination of ABT-263 with cytotoxic agents demonstrated superior tumor growth inhibition and delay in multiple models versus cytotoxic therapy alone, along with significant improvements in tumor response rates., Conclusions: Inhibition of the Bcl-2 family of proteins by ABT-263 enhances the cytotoxicity of multiple chemotherapeutics in hematologic tumors and represents a promising addition to the therapeutic arsenal for treatment of these diseases.

ABT-263 inhibits the antiapoptotic proteins Bcl-2, Bcl-x(L), and Bcl-w and has single-agent efficacy in numerous small cell lung carcinoma (SCLC) and leukemia/lymphoma cell lines in vitro and in vivo. It is currently in clinical trials for treating patients with SCLC and various leukemia/lymphomas. Identification of predictive markers for response will benefit the clinical development of ABT-263. We identified the expression of Bcl-2 family genes that correlated best with sensitivity to ABT-263 in a panel of 36 SCLC and 31 leukemia/lymphoma cell lines. In cells sensitive to ABT-263, expression of Bcl-2 and Noxa is elevated, whereas expression of Mcl-1 is higher in resistant cells. We also examined global expression differences to identify gene signature sets that correlated with sensitivity to ABT-263 to generate optimal signature sets predictive of sensitivity to ABT-263. Independent cell lines were used to verify the predictive power of the gene sets and to refine the optimal gene signatures. When comparing normal lung tissue and SCLC primary tumors, the expression pattern of these genes in the tumor tissue is most similar to sensitive SCLC lines, whereas normal tissue is most similar to resistant SCLC lines. Most of the genes identified using global expression patterns are related to the apoptotic pathway; however, all but Bcl-rambo are distinct from the Bcl-2 family. This study leverages global expression data to identify key gene expression patterns for sensitivity to ABT-263 in SCLC and leukemia/lymphoma and may provide guidance in the selection of patients in future clinical trials.

The Bcl-2 family of proteins plays a critical role in controlling immune responses by regulating the expansion and contraction of activated lymphocyte clones by apoptosis. ABT-737, which was originally developed for oncology, is a potent inhibitor of Bcl-2, Bcl-x(L), and Bcl-w protein function. There is evidence that Bcl-2-associated dysregulation of lymphocyte apoptosis may contribute to the pathogenesis of autoimmunity and lead to the development of autoimmune diseases. In this study, we report that ABT-737 treatment resulted in potent inhibition of lymphocyte proliferation as measured by in vitro mitogenic or ex vivo Ag-specific stimulation. More importantly, ABT-737 significantly reduced disease severity in tissue-specific and systemic animal models of autoimmunity. Bcl-2 family antagonism by ABT-737 was efficacious in treating animal models of arthritis and lupus. Our results suggest that treatment with a Bcl-2 family antagonist represents a novel and potentially attractive therapeutic approach for the clinical treatment of autoimmunity.

Overexpression of prosurvival proteins such as Bcl-2 and Bcl-X L has been correlated with tumorigenesis and resistance to chemotherapy, and thus, the development of antagonists of these proteins may provide a novel means for the treatment of cancer. We recently described the discovery of 1 (ABT-737), which binds Bcl-2, Bcl-X L, and Bcl-w with high affinity, shows robust antitumor activity in murine tumor xenograft models, but is not orally bioavailable. Herein, we report that targeted modifications at three key positions of 1 resulted in a 20-fold improvement in the pharmacokinetic/pharmacodynamic relationship (PK/PD) between oral exposure (AUC) and in vitro efficacy in human tumor cell lines (EC 50). The resulting compound, 2 (ABT-263), is orally efficacious in an established xenograft model of human small cell lung cancer, inducing complete tumor regressions in all animals. Compound 2 is currently in multiple phase 1 clinical trials in patients with small cell lung cancer and hematological malignancies.

ABT-263 is a potent, orally bioavailable inhibitor of the antiapoptotic Bcl-2 family members Bcl-2, Bcl-x(L), and Bcl-w, which is currently in phase I clinical trials. Previous work has shown that this compound has low nanomolar cell-killing activity in a variety of lymphoma and leukemia cell lines, many of which overexpress Bcl-2 through a variety of mechanisms. Rapamycin is a macrolide antibiotic that inhibits the mammalian target of rapamycin complex, leading to cell cycle arrest and inhibition of protein translation. Rapamycin (and its analogues) has shown activity in a variety of tumor cell lines primarily through induction of cell cycle arrest. Activity has also been shown clinically in mantle cell lymphoma and advanced renal cell carcinoma. Here, we show that treatment of the follicular lymphoma lines DoHH-2 and SuDHL-4 with 100 nmol/L rapamycin induces substantial G(0)-G(1) arrest. Addition of as little as 39 nmol/L ABT-263 to the rapamycin regimen induced a 3-fold increase in sub-G(0) cells. Combination of these agents also led to a significant increase in Annexin V staining over ABT-263 alone. In xenograft models of these tumors, rapamycin induced a largely cytostatic response in the DoHH-2 and SuDHL-4 models. Coadministration with ABT-263 induced significant tumor regression, with DoHH-2 and SuDHL-4 tumors showing 100% overall response rates. Apoptosis in these tumors was significantly enhanced by combination therapy as measured by staining with an antibody specific for cleaved caspase-3. These data suggest that combination of ABT-263 and rapamycin or its analogues represents a promising therapeutic strategy for the treatment of lymphoma.

Purpose: The purpose of this study was to characterize the activity of the Bcl-2 protein family inhibitor ABT-263 in a panel of small cell lung cancer (SCLC) xenograft models., Experimental Design: A panel of 11 SCLC xenograft models was established to evaluate the efficacy of ABT-263. Single agent activity was examined on a continuous dosing schedule in each of these models. The H146 model was used to further evaluate dose and schedule, comparison to standard cytotoxic agents, and induction of apoptosis., Results: ABT-263 exhibited a range of antitumor activity, leading to complete tumor regression in several models. Significant regressions of tumors as large as 1 cc were also observed. The efficacy of ABT-263 was also quite durable; in several cases, minimal tumor regrowth was noted several weeks after the cessation of treatment. Antitumor effects were equal or superior to that of several clinically approved cytotoxic agents. Regression of large established tumors was observed through several cycles of therapy and efficacy was retained in a Pgp-1 overexpressing line. Significant efficacy was observed on several dose and therapeutic schedules and was associated with significant induction of apoptosis., Conclusions: ABT-263 is a potent, orally bioavailable inhibitor of Bcl-2 family proteins that has recently entered clinical trials. The efficacy data reported here suggest that SCLC is a promising area of clinical investigation with this agent.

Overexpression of the prosurvival Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1) is commonly associated with tumor maintenance, progression, and chemoresistance. We previously reported the discovery of ABT-737, a potent, small-molecule Bcl-2 family protein inhibitor. A major limitation of ABT-737 is that it is not orally bioavailable, which would limit chronic single agent therapy and flexibility to dose in combination regimens. Here we report the biological properties of ABT-263, a potent, orally bioavailable Bad-like BH3 mimetic (K(i)'s of <1 nmol/L for Bcl-2, Bcl-xL, and Bcl-w). The oral bioavailability of ABT-263 in preclinical animal models is 20% to 50%, depending on formulation. ABT-263 disrupts Bcl-2/Bcl-xL interactions with pro-death proteins (e.g., Bim), leading to the initiation of apoptosis within 2 hours posttreatment. In human tumor cells, ABT-263 induces Bax translocation, cytochrome c release, and subsequent apoptosis. Oral administration of ABT-263 alone induces complete tumor regressions in xenograft models of small-cell lung cancer and acute lymphoblastic leukemia. In xenograft models of aggressive B-cell lymphoma and multiple myeloma where ABT-263 exhibits modest or no single agent activity, it significantly enhances the efficacy of clinically relevant therapeutic regimens. These data provide the rationale for clinical trials evaluating ABT-263 in small-cell lung cancer and B-cell malignancies. The oral efficacy of ABT-263 should provide dosing flexibility to maximize clinical utility both as a single agent and in combination regimens.

The molecular chaperone HSP90 has been shown to facilitate cancer cell survival by stabilizing key proteins responsible for a malignant phenotype. We report here the results of parallel fragment-based drug design approaches in the design of novel HSP90 inhibitors. Initial aminopyrimidine leads were elaborated using high-throughput organic synthesis to yield nanomolar inhibitors of the enzyme. Second site leads were also identified which bound to HSP90 in two distinct conformations, an 'open' and 'closed' form. Intriguingly, linked fragment approaches targeting both of these conformations were successful in producing novel, micromolar inhibitors. Overall, this study shows that, with only a few fragment hits, multiple lead series can be generated for HSP90 due to the inherent flexibility of the active site. Thus, ample opportunities exist to use these lead series in the development of clinically useful HSP90 inhibitors for the treatment of cancers.

Survivin is one of the most tumor-specific genes in the human genome and is an attractive target for cancer therapy. However, small-molecule ligands for survivin have not yet been described. Thus, an interrogation of survivin which could potentially both validate a small-molecule therapy approach, and determine the biochemical nature of any of survivin's functions has not been possible. Here we describe the discovery and characterization of a small molecule binding site on the survivin surface distinct from the Smac peptide-binding site. The new site is located at the dimer interface and exhibits many of the features of highly druggable, biologically relevant protein binding sites. A variety of small hydrophobic compounds were found that bind with moderate affinity to this binding site, from which one lead was developed into a group of compounds with nanomolar affinity. Additionally, a subset of these compounds are adequately water-soluble and cell-permeable. Thus, the structural studies and small molecules described here provide tools that can be used to probe the biochemical role(s) of survivin, and may ultimately serve as a basis for the development of small molecule therapeutics acting via direct or allosteric disruption of binding events related to this poorly understood target.

Platelets are relatively short-lived, anucleated cells that are essential for proper hemostasis. The regulation of platelet survival in the circulation remains poorly understood. The process of platelet activation and senescence in vivo is associated with processes similar to those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, and cell shrinkage. ABT-737, a potent antagonist of Bcl-2, Bcl-X(L), and Bcl-w, induces apoptosis in nucleated cells dependent on these proteins for survival. In vivo, ABT-737 induces a reduction of circulating platelets that is maintained during drug therapy, followed by recovery to normal levels within several days after treatment cessation. Whole body scintography utilizing ([111])Indium-labeled platelets in dogs shows that ABT-737-induced platelet clearance is primarily mediated by the liver. In vitro, ABT-737 treatment leads to activation of key apoptotic processes including cytochrome c release, caspase-3 activation, and PS externalization in isolated platelets. Despite these changes, ABT-737 is ineffective in promoting platelet activation as measured by granule release markers and platelet aggregation. Taken together, these data suggest that ABT-737 induces an apoptosis-like response in platelets that is distinct from platelet activation and results in enhanced clearance in vivo by the reticuloendothelial system.

Overexpression of the antiapototic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional chemotherapy. Inhibition of these prosurvival proteins is an attractive strategy for cancer therapy. We recently described the discovery of a selective Bcl-xL antagonist that potentiates the antitumor activity of chemotherapy and radiation. Here we describe the use of structure-guided design to exploit a deep hydrophobic binding pocket on the surface of these proteins to develop the first dual, subnanomolar inhibitors of Bcl-xL and Bcl-2. This study culminated in the identification of 2, which exhibited EC50 values of 8 nM and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Compound 2 demonstrated single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2, and efficacy in a murine xenograft model of lymphoma when given both as a single agent and in combination with etoposide.

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.

One of the primary objectives in the design of protein inhibitors is to shape the three-dimensional structures of small molecules to be complementary to the binding site of a target protein. In the course of our efforts to discover potent inhibitors of Bcl-2 family proteins, we found a unique folded conformation adopted by tethered aromatic groups in the ligand that significantly enhanced binding affinity to Bcl-XL. This finding led us to design compounds that were biased by nonbonding interactions present in a urea tether to adopt this bioactive, folded motif. To characterize the key interactions that induce the desired conformational bias, a series of substituted N,N'-diarylureas were prepared and analyzed using X-ray crystallography and quantum mechanical calculations. Stabilizing pi-stacking interactions and destabilizing steric interactions were predicted to work in concert in two of the substitution patterns to promote the bioactive conformation as a global energy minimum and result in a high target binding affinity. Conversely, intramolecular hydrogen bonding present in the third substitution motif promotes a less active, extended conformer as the energetically favored geometry. These findings were corroborated when the inhibition constant of binding to Bcl-XL was determined for fully elaborated analogues bearing these structural motifs. Finally, we obtained the NMR solution structure of the disubstituted N,N'-diarylurea bound to Bcl-XL demonstrating the folded conformation of the urea motif engaged in extensive pi-interactions with the protein.

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-X(L) versus Bcl-2 (K(i)'s of 0.80 and 67 nmol/L for Bcl-X(L) and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC(50) of <500 nmol/L in cells dependent on Bcl-X(L) for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non-small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy.

Development of a rationally designed potentiator of cancer chemotherapy, via inhibition of Bcl-X(L) function, is described. Lead compounds generated by NMR screening and directed parallel synthesis displayed sub-microM binding but were strongly deactivated in the presence of serum. The dominant component of serum deactivation was identified as domain III of human serum albumin (HSA); NMR solution structures of inhibitors bound to both Bcl-X(L) and HSA domain III indicated two potential optimization sites for separation of affinities. Modifications at both sites resulted in compounds with improved Bcl-X(L) binding and greatly increased activity in the presence of human serum, culminating in 73R, which bound to Bcl-X(L) with a K(i) of 0.8 nM. In a cellular assay 73R reversed the protection afforded by Bcl-X(L) overexpression against cytokine deprivation in FL5.12 cells with an EC(50) of 0.47 microM. 73R showed little effect on the viability of the human non small cell lung cancer cell line A549. However, consistent with the proposed mechanism, 73R potentiated the activity of paclitaxel and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in a mouse xenograft model.

The antiapoptotic proteins Bcl-x(L) and Bcl-2 play key roles in the maintenance of normal cellular homeostasis. However, their overexpression can lead to oncogenic transformation and is responsible for drug resistance in certain types of cancer. This makes Bcl-x(L) and Bcl-2 attractive targets for the development of potential anticancer agents. Here we describe the structure-based discovery of a potent Bcl-x(L) inhibitor directed at a hydrophobic groove on the surface of the protein. This groove represents the binding site for BH3 peptides from proapoptotic Bcl-2 family members such as Bak and Bad. Application of NMR-based screening yielded an initial biaryl acid with an affinity (K(d)) of approximately 300 microM for the protein. Following the classical "SAR by NMR" approach, a second-site ligand was identified that bound proximal to the first-site ligand in the hydrophobic groove. From NMR-based structural studies and parallel synthesis, a potent ligand was obtained, which binds to Bcl-x(L) with an inhibition constant (K(i)) of 36 +/- 2 nM.

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

Non-peptidic small molecule SMAC mimetics were designed and synthesized that bind to the BIR3 domain of XIAP using structure-based design. Substituted five-membered heterocycles such as thiazoles and imidazoles were identified that serve as replacements for peptide fragments of the lead.

Inhibitor of apoptosis (IAP) proteins are overexpressed in many cancers and have been implicated in tumor growth, pathogenesis, and resistance to chemo- or radiotherapy. On the basis of the NMR structure of a SMAC peptide complexed with the BIR3 domain of X-linked IAP (XIAP), a novel series of XIAP antagonists was discovered. The most potent compounds in this series bind to the baculovirus IAP repeat 3 (BIR3) domain of XIAP with single-digit nanomolar affinity and promote cell death in several human cancer cell lines. In a MDA-MB-231 breast cancer mouse xenograft model, these XIAP antagonists inhibited the growth of tumors. Close structural analogues that showed only weak binding to the XIAP-BIR3 domain were inactive in the cellular assays and showed only marginal in vivo activity. Our results are consistent with a mechanism in which ligands for the BIR3 domain of XIAP induce apoptosis by freeing up caspases. The present study validates the BIR3 domain of XIAP as a target and supports the use of small molecule XIAP antagonists as a potential therapy for cancers that overexpress XIAP.

The SAR at C-5 of the 10-methoxy-2,2,4-trimethylbenzopyrano[3,4-f]quinoline core leading to identification of (-) anti 1-methylcyclohexen-3-yl as the optimum substituent that imparts minimal GR mediated in vitro transcriptional activation while maintaining full transcriptional repression is described. The in vitro profile of these candidates in human cell assays relevant to the therapeutic window of glucocorticoid modulators is outlined.

Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor gamma coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.

The preparation and characterization of a series of C-10 substituted 5-allyl-2,5-dihydro-2,2,4-trimethyl-1H-[1]benzopyrano[3,4-f]quinolines as a novel class of selective ligands for the glucocorticoid receptor is described. Substitution at the C-10 position of the tetracyclic core with linear, two-atom appendages (OCH(3), OCF(2)H, NHMe, SMe, CH=CH(2), Ctbd1;CH, CH(2)OH) provided molecules of high affinity (K(i) = 2-8 nM) for the human glucocorticoid receptor (hGR) with limited cross-reactivity with other steroid receptors (PR, MR, AR, ER). Optimal analogues showed slightly less potent but highly efficacious E-selectin repression with reduced levels of GRE activation efficacy in reporter gene assays relative to prednisolone. Preliminary SAR of analogues containing substitution at the C-9 and C-10 positions identified the 9-OH, 10-OMe analogue 50 and the 9-OH, 10-Cl analogue 58 as compounds that demonstrated potent, GR-mediated inhibition in a conconavalin A stimulated T-cell proliferation assay in both rodent and human whole blood monocytes. When evaluated for their in vivo effects in carrageenan-induced paw edema in rats, 50, 58, and 10-OCF(2)H analogue 35 showed dose-dependent anti-inflammatory effects (50, ED(50) = 16 mg/kg; 58, ED(50) = 15 mg/kg; 35, ED(50) = 21 mg/kg vs ED(50) = 15 mg/kg for 18 and ED(50) = 4 mg/kg for prednisolone).

Glucocorticoids have a pervasive role in human health and physiology. The endogenous members of this family are involved in a breadth of endocrine functions including metabolism of lipids, carbohydrates and proteins, stress response, fluid and electrolyte balance, as well as maintenance of immunological, renal and skeletal homeostasis. The predominant mode of action of glucocorticoids involves regulation of gene expression via the glucocorticoid receptor (GR). Synthetic glucocorticoids have long been the standard for the treatment of inflammatory and immune disorders, yet the benefits of classic steroids such as dexamethasone and prednisolone are accompanied by well-characterized potentiation of homeostatic endocrine functions, leading to the side effects associated with prolonged treatment. In recent campaigns for safer analogs, compounds have been sought which differentiate functional repression of existing transcription factors such as AP-1 and NFkappaB from GR-mediated transcriptional activation arising from binding at glucocorticoid-receptor response elements (GREs). Such differentiated ligands would provide the desired immunoregulatory actions without the endogenous changes in gene expression associated with undifferentiated steroids. We detail the methods for the evaluation of selective GR modulators and describe the evolution of new compounds where varying degrees of selectivity have been reported.

The preparation and characterization of a series of selective glucocorticoid receptor modulators are described. The preliminary structure-activity relationship of nonaromatic C-5 substitution on the tetracyclic quinoline core showed a preference for small lipophilic side chains. Proper substitution at this position maintained the transcriptional repression of proinflammatory transcription factors while diminishing the transcriptional activation activity of the ligand/glucocorticoid receptor complex. The optimal compounds described in this study were the allyl analogue 18 and cyclopentyl analogue 32. These candidates showed slightly less potent, highly efficacious E-selectin repression with significantly reduced levels of glucocorticoid response element activation in reporter gene assays vs prednisolone. Allyl analogue 18 was evaluated in vivo. An oral dose of 18 showed an ED(50) = 1.7 mg/kg as compared to 1.2 mg/kg for prednisolone in the Sephadex-induced pulmonary eosinophilia model and an ED(50) = 15 mg/kg vs 4 mg/kg for prednisolone in the carrageenan-induced paw edema model.

A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (K(i) = 2.4-9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED(50) = 2.8 mpk for 13 vs ED(50) = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).

In search of a uroselective agent that exhibits a high level of selectivity for the alpha(1A) receptor, a novel series of tricyclic hexahydrobenz[e]isoindoles was synthesized. A generic pharmacophoric model was developed requiring the presence of a basic amine core and a fused heterocyclic side chain separated by an alkyl chain. It was shown that the 6-OMe substitution with R, R stereochemistry of the ring junction of the benz[e]isoindole and a two-carbon spacer chain were optimal. In contrast to the highly specific requirements for the benz[e]isoindole portion and linker chain, a wide variety of tricyclic fused heterocyclic attachments were tolerated with retention of potency and selectivity. In vitro functional assays for the alpha(1) adrenoceptor subtypes were used to further characterize these compounds, and in vivo models of vascular vs prostatic tone were used to assess uroselectivity.