Structure-Based Design of A-1293102, a Potent and Selective BCL-XL Inhibitor.

BCL-XL, 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-XL inhibitors exemplified by A-1293102. This molecule contains structural elements of selective BCL-XL inhibitor A-1155463 and the dual BCL-XL/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-XL and both efficiently and selectively killed BCL-XL-dependent tumor cells. X-ray crystallographic analysis demonstrated a key hydrogen bonding network in the P2 binding pocket of BCL-XL, 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-XL inhibitors, and thus serves as a useful tool for biological studies as well as a lead compound for further optimization.

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.

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-XL Inhibitor.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. 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.

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.

lncRNA-MIAT regulates microvascular dysfunction by functioning as a competing endogenous RNA.

RATIONALE: Pathological angiogenesis is a critical component of diseases, such as ocular disorders, cancers, and atherosclerosis. It is usually caused by the abnormal activity of biological processes, such as cell proliferation, cell motility, immune, or inflammation response. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of these biological processes. However, the role of lncRNA in diabetes mellitus-induced microvascular dysfunction is largely unknown. OBJECTIVE: To elucidate whether lncRNA-myocardial infarction-associated transcript (MIAT) is involved in diabetes mellitus-induced microvascular dysfunction. METHODS AND RESULTS: Using quantitative polymerase chain reaction, we demonstrated increased expression of lncRNA-MIAT in diabetic retinas and endothelial cells cultured in high glucose medium. Visual electrophysiology examination, TUNEL staining, retinal trypsin digestion, vascular permeability assay, and in vitro studies revealed that MIAT knockdown obviously ameliorated diabetes mellitus-induced retinal microvascular dysfunction in vivo, and inhibited endothelial cell proliferation, migration, and tube formation in vitro. Bioinformatics analysis, luciferase assay, RNA immunoprecipitation, and in vitro studies revealed that MIAT functioned as a competing endogenous RNA, and formed a feedback loop with vascular endothelial growth factor and miR-150-5p to regulate endothelial cell function. CONCLUSIONS: This study highlights the involvement of lncRNA-MIAT in pathological angiogenesis and facilitates the development of lncRNA-directed diagnostics and therapeutics against neovascular diseases.

Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity.

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.

Structure-Guided Rescaffolding of Selective Antagonists of BCL-XL.

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.

Regulation of autophagy by high glucose in human retinal pigment epithelium.

BACKGROUND: Autophagy is a self-degradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress. Retinal pigment epithelium (RPE) works as the outer blood retina barrier and is vulnerable to energy stress-induced injury. However, the effect of high glucose treatment on autophagy is still unclear in RPE. METHODS: Transmission electron microscopy was used to detect the generation of autophagosome. Small interfering RNA (siRNA) and MTT was used to determine the effect of autophagy on cell viability. Western blots and immunohistochemistry were used to detect the expression pattern of autophagic markers, including LC3 and p62. RESULTS: High glucose treatment results in a significant increase in the generation of autophagosome and altered expression of LC3 and p62. High glucose-induced autophagy is independent of mTOR signaling, but is mainly regulated via ROS-mediated ER stress signaling. CONCLUSION: In the scenario of high glucose-induced oxidative stress, autophagy may be required for the removal of damaged proteins, and provide a default mechanism to prevent high glucose-induced injury in RPE.

Aberrant expression of long noncoding RNAs in early diabetic retinopathy.

PURPOSE: Long noncoding RNAs (lncRNAs) are broadly classified as transcripts longer than 200 nucleotides. lncRNA-mediated biology has been implicated in a variety of cellular processes and human diseases. Diabetic retinopathy (DR) is one of the leading causes of blindness. However, little is known about the role of lncRNAs in DR The goal of this study aimed to identify lncRNAs involved in early DR and characterize their roles in DR pathogenesis. METHODS: We established a mouse model of streptozotocin (STZ)-induced diabetes, and performed lncRNA expression profiling of retinas using microarray analysis. Based on the Pearson correlation analysis, an lncRNA/mRNA coexpression network was constructed. Gene ontology (GO) enrichment and KEGG analysis of lncRNAs-coexpressed mRNAs was conducted to identify the related biological modules and pathologic pathways. Real-time PCR was conducted to detect the expression pattern of lncRNA in the clinical samples and the RF/6A cell model of hyperglycemia. RESULTS: Approximately 303 lncRNAs were aberrantly expressed in the retinas of early DR, including 214 downregulated lncRNAs and 89 upregulated lncRNAs. GO analysis indicated that these lncRNAs-coexpressed mRNAs were targeted to eye development process (ontology: biological process), integral to membrane (ontology: cellular component), and structural molecule activity (ontology: molecular function). Pathway analysis indicated that lncRNAs-coexpressed mRNAs were mostly enriched in axon guidance signaling pathway. In addition, MALAT1, a conserved lncRNA, was significantly upregulated in an RF/6A cell model of hyperglycemia, in the aqueous humor samples, and in fibrovascular membranes of diabetic patients. CONCLUSIONS: lncRNAs are involved in the pathogenesis of DR through the modulation of multiple pathogenetic pathways. MALAT1, a conserved lncRNA, may become a potential therapeutic target for the prognosis, diagnosis, and treatment of DR.

Epigenetics and ocular diseases: from basic biology to clinical study.

Epigenetics is an emerging field in ophthalmology and has opened a new avenue for understanding ocular development and ocular diseases related to aging and environment. Epigenetic mechanisms, including DNA methylation, histone modifications, chromatin remodeling, and deployment of non-coding RNAs, result in the heritable silencing of gene expression without any change in DNA sequence. Accumulating evidence suggests a potential link between gene expression, chromatin structure, non-coding RNAs, and cellular differentiation during ocular development. Disruption of the balance of epigenetic networks could become the etiology of several ocular diseases. Here, we summarized the current knowledge about epigenetic regulatory mechanisms in ocular development and diseases.

Epigallocatechin-gallate (EGCG) regulates autophagy in human retinal pigment epithelial cells: a potential role for reducing UVB light-induced retinal damage.

Autophagy is an intracellular catabolic process involved in protein and organelle degradation via the lysosomal pathway that has been linked in the pathogenesis of age-related macular degeneration (AMD). UVB irradiation-mediated degeneration of the macular retinal pigment epithelial (RPE) cells is an important hallmark of AMD, which is along with the change in RPE autophagy. Thus, pharmacological manipulation of RPE autophagy may offer an alternative therapeutic target in AMD. Here, we found that epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, plays a regulatory role in UVB irradiation-induced autophagy in RPE cells. UVB irradiation results in a marked increase in the amount of LC3-II protein in a dose-dependent manner. EGCG administration leads to a significant reduction in the formation of LC3-II and autophagosomes. mTOR signaling activation is required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation is significantly impaired by rapamycin administration. Moreover, EGCG significantly alleviates the toxic effects of UVB irradiation on RPE cells in an autophagy-dependent manner. Collectively, our study reveals a novel role of EGCG in RPE autophagy. EGCG may be exploited as a potential therapeutic reagent for the treatment of pathological conditions associated with abnormal autophagy.

The electromagnetic-trait imaging computation of traveling wave method in breast tumor microwave sensor system.

Using the difference of dielectric constant between malignant tumor tissue and normal breast tissue, breast tumor microwave sensor system (BRATUMASS) determines the detected target of imaging electromagnetic trait by analyzing the properties of target tissue back wave obtained after near-field microwave radicalization (conelrad). The key of obtained target properties relationship and reconstructed detected space is to analyze the characteristics of the whole process from microwave transmission to back wave reception. Using traveling wave method, we derive spatial transmission properties and the relationship of the relation detected points distances, and valuate the properties of each unit by statistical valuation theory. This chapter gives the experimental data analysis results.

Discovery of 3H-benzo[4,5]thieno[3,2-d]pyrimidin-4-ones as potent, highly selective, and orally bioavailable inhibitors of the human protooncogene proviral insertion site in moloney murine leukemia virus (PIM) kinases.

Pim-1, Pim-2, and Pim-3 are a family of serine/threonine kinases which have been found to be overexpressed in a variety of hematopoietic malignancies and solid tumors. Benzothienopyrimidinones were discovered as a novel class of Pim inhibitors that potently inhibit all three Pim kinases with subnanomolar to low single-digit nanomolar K(i) values and exhibit excellent selectivity against a panel of diverse kinases. Protein crystal structures of the bound Pim-1 complexes of benzothienopyrimidinones 3b (PDB code 3JYA), 6e (PDB code 3JYO), and 12b (PDB code 3JXW) were determined and used to guide SAR studies. Multiple compounds exhibited potent antiproliferative activity in K562 and MV4-11 cells with submicromolar EC(50) values. For example, compound 14j inhibited the growth of K562 cells with an EC(50) value of 1.7 muM and showed K(i) values of 2, 3, and 0.5 nM against Pim-1, Pim-2, and Pim-3, respectively. These novel Pim kinase inhibitors efficiently interrupted the phosphorylation of Bad in both K562 and LnCaP-Bad cell lines, indicating that their potent biological activities are mechanism-based. The pharmacokinetics of 14j was studied in CD-1 mice and shown to exhibit bioavailability of 76% after oral dosing. ADME profiling of 14j suggested a long half-life in both human and mouse liver microsomes, good permeability, modest protein binding, and no CYP inhibition below 20 muM concentration.

Cyclin B1 is an efficacy-predicting biomarker for Chk1 inhibitors.

Chk1 is the major mediator of cell-cycle checkpoints in response to various forms of genotoxic stress. Although it was previously speculated that checkpoint abrogation due to Chk1 inhibition may potentiate the efficacy of DNA-damaging agents through induction of mitotic catastrophe, there has not been direct evidence proving this process. Here, through both molecular marker and morphological analysis, we directly demonstrate that specific downregulation of Chk1 expression by Chk1 siRNA potentiates the cytotoxicities of topoisomerase inhibitors through the induction of premature chromosomal condensation and mitotic catastrophe. More importantly, we discovered that the cellular cyclin B1 level is the major determinant of the potentiation. We show that downregulation of cyclin B1 leads to impairment of the induction of mitotic catastrophe and correspondingly a reduction of the potentiation ability of either Chk1 siRNA or a small molecule Chk1 inhibitor. More significantly, we have extended the study by examining a panel of 10 cancer cell-lines with different tissue origins for their endogenous levels of cyclin B1 and the ability of a Chk1 inhibitor to sensitize the cells to DNA-damaging agents. The cellular levels of cyclin B1 positively correlate with the degrees of potentiation achieved. Of additional interest, we observed that the various colon cancer cell lines in general appear to express higher levels of cyclin B1 and also display higher sensitivity to Chk1 inhibitors, implying that Chk1 inhibitor may be more efficacious in treating colon cancers. In summary, we propose that cyclin B1 is a biomarker predictive of the efficacy of Chk1 inhibitors across different types of cancers. Unlike previously established efficacy-predictive biomarkers that are usually the direct targets of the therapeutic agents, cyclin B1 represents a non-drug-target biomarker that is based on the mechanism of action of the target inhibitor. This finding may be potentially very useful for the stratification of patients for Chk1 inhibitor clinical trials and hence, maximize its chance of success.

Macrocyclic ureas as potent and selective Chk1 inhibitors: an improved synthesis, kinome profiling, structure-activity relationships, and preliminary pharmacokinetics.

A new series of potent macrocyclic urea-based Chk1 inhibitors are described. A detailed SAR study on the 4-position of the phenyl ring of the 14-member macrocyclic ureas 1a and d led to the identification of the potent Chk1 inhibitors 2, 5-7, 10, 13, 14, 19-21, 25, 27, and 31-34. These compounds significantly sensitize tumor cells to the DNA-damaging antitumor agent doxorubicin in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and camptothecin-induced S checkpoints, indicating that the potent biological activities of these compounds are mechanism-based through Chk1 inhibition. Kinome profiling analysis of a representative macrocyclic urea 25 against a panel of 120 kinases indicates that these novel macrocyclic ureas are highly selective Chk1 inhibitors. Preliminary PK studies of 1a and b suggest that the 14-member macrocyclic inhibitors may possess better PK properties than their 15-member counterparts. An improved synthesis of 2 and 20 by using 2-(trimethylsilyl)ethoxycarbonyl (Teoc) to protect the amino group not only readily provided the desired compounds in pure form but also facilitated the scale up of potent compounds for various biological studies.

Synthesis and in-vitro biological activity of macrocyclic urea Chk1 inhibitors.

A variety of macrocyclic urea compounds were prepared as potent Chk1 inhibitors by modifying the C5 position of the benzene ring of the macrocyclic urea with ether moieties, aliphatic carbon chains, amide and halides. Enzymatic activity less than 20nM was observed in 29 of 40 compounds. Compounds 14, 46d, and 48j provided the best overall results in the cellular assays as they abrogated doxorubicin-induced cell cycle arrest (IC(50)=3.31, 3.08, and 3.13microM) and enhanced doxorubicin cytotoxicity (IC(50)=0.54, 1.27, and 0.96microM) while displaying no single agent activity, respectively.

Cyanopyridyl containing 1,4-dihydroindeno[1,2-c]pyrazoles as potent checkpoint kinase 1 inhibitors: improving oral biovailability.

A series of 1,4-dihydroindeno[1,2-c]pyrazole compounds with a cyanopyridine moiety at the 3-position of the tricyclic pyrazole core was explored as potent CHK-1 inhibitors. The impact of substitutions at the 6 and/or 7-position of the core on pharmacokinetic properties was studied in detail. Compounds carrying a side chain with an ether linker at the 7-position and a terminal morpholino group, such as 29 and 30, exhibited much-improved oral biovailability in mice as compared to earlier generation inhibitors. These compounds also possessed desirable cellular activity in potentiating doxorubicin and will serve as valuable tool compounds for in vivo evaluation of CHK-1 inhibitors to sensitize DNA-damaging agents.