We report herein the discovery of highly potent PROTAC degraders of androgen receptor (AR), as exemplified by compound 34 (ARD-69). ARD-69 induces degradation of AR protein in AR-positive prostate cancer cell lines in a dose- and time-dependent manner. ARD-69 achieves DC50 values of 0.86, 0.76, and 10.4 nM in LNCaP, VCaP, and 22Rv1 AR+ prostate cancer cell lines, respectively. ARD-69 is capable of reducing the AR protein level by >95% in these prostate cancer cell lines and effectively suppressing AR-regulated gene expression. ARD-69 potently inhibits cell growth in these AR-positive prostate cancer cell lines and is >100 times more potent than AR antagonists. A single dose of ARD-69 effectively reduces the level of AR protein in xenograft tumor tissue in mice. Further optimization of ARD-69 may ultimately lead to a new therapy for AR+, castration-resistant prostate cancer.

The bromodomain and extra-terminal (BET) family proteins, consisting of BRD2, BRD3, BRD4, and testis-specific BRDT members, are epigenetic “readers” and play a key role in the regulation of gene transcription. BET proteins are considered to be attractive therapeutic targets for cancer and other human diseases. Recently, heterobifunctional small-molecule BET degraders have been designed based upon the proteolysis targeting chimera (PROTAC) concept to induce BET protein degradation. Herein, we present our design, synthesis, and evaluation of a new class of PROTAC BET degraders. One of the most promising compounds, 23, effectively degrades BRD4 protein at concentrations as low as 30 pM in the RS4;11 leukemia cell line, achieves an IC50 value of 51 pM in inhibition of RS4;11 cell growth and induces rapid tumor regression in vivo against RS4;11 xenograft tumors. These data establish that compound 23 (BETd-260/ZBC260) is a highly potent and efficacious BET degrader.

Proteins of the bromodomain and extra-terminal (BET) family are epigenetics "readers" and promising therapeutic targets for cancer and other human diseases. We describe herein a structure-guided design of [1,4]oxazepines as a new class of BET inhibitors and our subsequent design, synthesis, and evaluation of proteolysis-targeting chimeric (PROTAC) small-molecule BET degraders. Our efforts have led to the discovery of extremely potent BET degraders, exemplified by QCA570, which effectively induces degradation of BET proteins and inhibits cell growth in human acute leukemia cell lines even at low picomolar concentrations. QCA570 achieves complete and durable tumor regression in leukemia xenograft models in mice at well-tolerated dose-schedules. QCA570 is the most potent and efficacious BET degrader reported to date.

mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53-and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.

A novel chloride intracellular channel (CLIC) gene, clone mc3s5/mtCLIC, has been identified from differential display analysis of differentiating mouse keratinocytes from p53؉/؉ and p53؊/؊ mice. The 4.2-kilobase pair cDNA contains an open reading frame of 762 base pairs encoding a 253-amino acid protein with two putative transmembrane domains. mc3s5/mtCLIC protein shares extensive homology with a family of intracellular organelle chloride channels but is the first shown to be differentially regulated. mc3s5/mtCLIC mRNA is expressed to the greatest extent in vivo in heart, lung, liver, kidney, and skin, with reduced levels in some organs from p53؊/؊ mice. mc3s5/mtCLIC mRNA and protein are higher in p53؉/؉ compared with p53؊/؊ basal keratinocytes in culture, and both increase in differentiating keratinocytes independent of genotype. Overexpression of p53 in keratinocytes induces mc3s5/mtCLIC mRNA and protein. Exogenous human recombinant tumor necrosis factor ␣ also up-regulates mc3s5/mtCLIC mRNA and protein in keratinocytes. Subcellular fractionation of keratinocytes indicates that both the green fluorescent protein-mc3s5 fusion protein and the endogenous mc3s5/mtCLIC are localized to the cytoplasm and mitochondria. Similarly, mc3s5/mtCLIC was localized to mitochondria and cytoplasmic fractions of rat liver homogenates. Furthermore, mc3s5/mtCLIC colocalized with cytochrome oxidase in keratinocyte mitochondria by immunofluorescence and was also detected in the cytoplasmic compartment. Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a CLIC type chloride channel in mitochondria and the first indication that expression of an organellular chloride channel can be regulated by p53 and tumor necrosis factor ␣.The multiple functions now ascribed to the p53 tumor suppressor gene include cell cycle control, DNA repair, senescence, differentiation, and apoptosis (reviewed in Refs. 1-3). p53 exerts part of its biological function by activating or repressing the transcription of several target genes (2, 4, 5). p53 is particularly important in the skin because it is frequently mutated in preneoplastic and neoplastic human skin lesions (6), and loss of p53 enhances malignant progression of experimentally induced murine skin tumors (7). In keratinocytes, p53-dependent transcriptional activity increases in association with terminal differentiation (8). However, the skin of p53-deficient mice appears to be normal in vivo (9), and the expression of differentiation markers by p53Ϫ/Ϫ keratinocytes in vitro cannot be distinguished from that of wild-type (8). This implies that p53 is dispensable for normal skin development and differentiation. However, deletion of one or both p53 alleles enhances the establishment of immortal keratinocyte cell lines in vitro (10), suggesting that p53 could participate in keratinocyte mortality in a more subtle way, not involving expression of the major differentiation-related markers. To detect such a con...

Triple-negative breast cancers (TNBC) remain clinically challenging with a lack of options for targeted therapy. In this study, we report the development of a second-generation BET bromodomain (BRD) inhibitor, BETd-246, which exhibits superior selectivity, potency and antitumor activity. In human TNBC cells, BETd-246 induced degradation of BET transcription factors at low nanomolar concentrations within 1 hr of exposure, resulting in robust growth inhibition and apoptosis. BETd-246 was more potent and effective in TNBC cells than its parental BET inhibitor compound BETi-211. RNA-seq analysis revealed predominant downregulation of a large number of genes involved in proliferation and apoptosis in cells treated with BETd-246, as compared to BETi-211 treatment which upregulated and downregulated a similar number of genes. Functional investigations identified the MCL1 gene as a critical downstream effector of these BET degraders, which synergized with small molecule inhibitors of BCL-xL in triggering apoptosis. In multiple murine xenograft models of human breast cancer, BETd-246 and a further optimized analogue BETd-260 effectively depleted BET proteins in tumors and exhibited strong antitumor activities at well-tolerated dosing schedules. Overall, our findings show how specific targeting of BET proteins for degradation yields an effective therapeutic strategy for TNBC treatment.

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-␣ nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis.

Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A-G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.