Discovery of AK-1690: A Potent and Highly Selective STAT6 PROTAC Degrader.

STAT6 is an attractive therapeutic target for human cancers and other human diseases. Starting from a STAT6 ligand with Ki = 3.5 µM binding affinity, we obtained AK-068 with Ki = 6 nM to STAT6 and at least >85-fold binding selectivity over STAT5. Using AK-068 and cereblon ligands, we discovered AK-1690 as the first, potent and selective PROTAC STAT6 degrader. AK-1690 effectively induces degradation of STAT6 protein in cells with DC50 values of as low as 1 nM while showing minimal effect on other STAT members up to 10 µM. A single dose of AK-1690 effectively depletes STAT6 in mouse tissues. Determination of the first cocrystal structure of STAT6 in complex with AK-1690 provides a structural basis for their interactions. AK-1690 is a powerful tool with which to investigate the roles of STAT6 in human diseases and biological processes and a promising lead compound for further optimization.

Discovery of CW-3308 as a Potent, Selective, and Orally Efficacious PROTAC Degrader of BRD9.

The bromodomain-containing protein BRD9 has emerged as an attractive therapeutic target. In the present study, we successfully identified a number of highly potent BRD9 degraders by using two different cereblon ligands developed in our laboratory. Further optimization led to the discovery of CW-3308 as a potent, selective, and orally bioavailable BRD9 degrader. It displayed degradation potency (DC50) < 10 nM and efficiency (Dmax) > 90% against BRD9 in the G401 rhabdoid tumor and HS-SY-II synovial sarcoma cell lines and had a high degradation selectivity over BRD7 and BRD4 proteins. CW-3308 achieved 91% of oral bioavailability in mice. A single oral dose efficiently reduced the BRD9 protein by >90% in the synovial sarcoma HS-SY-II xenograft tumor tissue. Oral administration effectively inhibited HS-SY-II xenograft tumor growth in mice. CW-3308 is a promising lead compound for further optimization and extensive evaluation for the treatment of synovial sarcoma, rhabdoid tumor, and other BRD9-dependent human diseases.

IL-4-STAT6 axis amplifies histamine-induced vascular endothelial dysfunction and hypovolemic shock.

BACKGROUND: Mast cell-derived mediators induce vasodilatation and fluid extravasation, leading to cardiovascular failure in severe anaphylaxis. We previously revealed a synergistic interaction between the cytokine IL-4 and the mast cell-derived mediator histamine in modulating vascular endothelial (VE) dysfunction and severe anaphylaxis. The mechanism by which IL-4 exacerbates histamine-induced VE dysfunction and severe anaphylaxis is unknown. OBJECTIVE: We sought to identify the IL-4-induced molecular processes regulating the amplification of histamine-induced VE barrier dysfunction and the severity of IgE-mediated anaphylactic reactions. METHODS: RNA sequencing, Western blot, Ca2+ imaging, and barrier functional analyses were performed on the VE cell line (EA.hy926). Pharmacologic degraders (selective proteolysis-targeting chimera) and genetic (lentiviral short hairpin RNA) inhibitors were used to determine the roles of signal transducer and activator of transcription 3 (STAT3) and STAT6 in conjunction with in vivo model systems of histamine-induced hypovolemic shock. RESULTS: IL-4 enhancement of histamine-induced VE barrier dysfunction was associated with increased VE-cadherin degradation, intracellular calcium flux, and phosphorylated Src levels and required transcription and de novo protein synthesis. RNA sequencing analyses of IL-4-stimulated VE cells identified dysregulation of genes involved in cell proliferation, cell development, and cell growth, and transcription factor motif analyses revealed a significant enrichment of differential expressed genes with putative STAT3 and STAT6 motif. IL-4 stimulation in EA.hy926 cells induced both serine residue 727 and tyrosine residue 705 phosphorylation of STAT3. Genetic and pharmacologic ablation of VE STAT3 activity revealed a role for STAT3 in basal VE barrier function; however, IL-4 enhancement and histamine-induced VE barrier dysfunction was predominantly STAT3 independent. In contrast, IL-4 enhancement and histamine-induced VE barrier dysfunction was STAT6 dependent. Consistent with this finding, pharmacologic knockdown of STAT6 abrogated IL-4-mediated amplification of histamine-induced hypovolemia. CONCLUSIONS: These studies unveil a novel role of the IL-4/STAT6 signaling axis in the priming of VE cells predisposing to exacerbation of histamine-induced anaphylaxis.

Discovery of CBPD-409 as a Highly Potent, Selective, and Orally Efficacious CBP/p300 PROTAC Degrader for the Treatment of Advanced Prostate Cancer.

CBP/p300 are critical transcriptional coactivators of the androgen receptor (AR) and are promising cancer therapeutic targets. Herein, we report the discovery of highly potent, selective, and orally bioavailable CBP/p300 degraders using the PROTAC technology with CBPD-409 being the most promising compound. CBPD-409 induces robust CBP/p300 degradation with DC50 0.2-0.4 nM and displays strong antiproliferative effects with IC50 1.2-2.0 nM in the VCaP, LNCaP, and 22Rv1 AR+ prostate cancer cell lines. It has a favorable pharmacokinetic profile and achieves 50% of oral bioavailability in mice. A single oral administration of CBPD-409 at 1 mg/kg achieves >95% depletion of CBP/p300 proteins in the VCaP tumor tissue. CBPD-409 exhibits strong tumor growth inhibition and is much more potent and efficacious than two CBP/p300 inhibitors CCS1477 and GNE-049 and the AR antagonist Enzalutamide. CBPD-409 is a promising CBP/p300 degrader for further extensive evaluations for the treatment of advanced prostate cancer and other types of human cancers.

Discovery of CBPD-268 as an Exceptionally Potent and Orally Efficacious CBP/p300 PROTAC Degrader Capable of Achieving Tumor Regression.

CBP/p300 proteins are key epigenetic regulators and promising targets for the treatment of castration-resistant prostate cancer and other types of human cancers. Herein, we report the discovery and characterization of CBPD-268 as an exceptionally potent, effective, and orally efficacious PROTAC degrader of CBP/p300 proteins. CBPD-268 induces CBP/p300 degradation in three androgen receptor-positive prostate cancer cell lines, with DC50 ≤ 0.03 nM and Dmax > 95%, leading to potent cell growth inhibition. It has an excellent oral bioavailability in mice and rats. Oral administration of CBPD-268 at 0.3-3 mg/kg resulted in profound and persistent CBP/p300 depletion in tumor tissues and achieved strong antitumor activity in the VCaP and 22Rv1 xenograft tumor models in mice, including tumor regression in the VCaP tumor model. CBPD-268 was well tolerated in mice and rats and displayed a therapeutic index of >10. Taking these results together, CBPD-268 is a highly promising CBP/p300 degrader as a potential new cancer therapy.

Discovery of ARD-1676 as a Highly Potent and Orally Efficacious AR PROTAC Degrader with a Broad Activity against AR Mutants for the Treatment of AR + Human Prostate Cancer.

We report herein the discovery and extensive characterization of ARD-1676, a highly potent and orally efficacious PROTAC degrader of the androgen receptor (AR). ARD-1676 was designed using a new class of AR ligands and a novel cereblon ligand. It has DC50 values of 0.1 and 1.1 nM in AR+ VCaP and LNCaP cell lines, respectively, and IC50 values of 11.5 and 2.8 nM in VCaP and LNCaP cell lines, respectively. ARD-1676 effectively induces degradation of a broad panel of clinically relevant AR mutants. ARD-1676 has an oral bioavailability of 67, 44, 31, and 99% in mice, rats, dogs, and monkeys, respectively. Oral administration of ARD-1676 effectively reduces the level of AR protein in the VCaP tumor tissue in mice and inhibits tumor growth in the VCaP mouse xenograft tumor model without any sign of toxicity. ARD-1676 is a highly promising development candidate for the treatment of AR+ human prostate cancer.

Discovery of ERD-3111 as a Potent and Orally Efficacious Estrogen Receptor PROTAC Degrader with Strong Antitumor Activity.

Estrogen receptor α (ERα) is a prime target for the treatment of ER-positive (ER+) breast cancer. Despite the development of several effective therapies targeting ERα signaling, clinical resistance remains a major challenge. In this study, we report the discovery of a new class of potent and orally bioavailable ERα degraders using the PROTAC technology, with ERD-3111 being the most promising compound. ERD-3111 exhibits potent in vitro degradation activity against ERα and demonstrates high oral bioavailability in mice, rats, and dogs. Oral administration of ERD-3111 effectively reduces the levels of wild-type and mutated ERα proteins in tumor tissues. ERD-3111 achieves tumor regression or complete tumor growth inhibition in the parental MCF-7 xenograft model with wild-type ER and two clinically relevant ESR1 mutated models in mice. ERD-3111 is a promising ERα degrader for further extensive evaluations for the treatment of ER+ breast cancer.

A selective small-molecule STAT5 PROTAC degrader capable of achieving tumor regression in vivo.

Signal transducer and activator of transcription 5 (STAT5) is an attractive therapeutic target, but successful targeting of STAT5 has proved to be difficult. Here we report the development of AK-2292 as a first, potent and selective small-molecule degrader of both STAT5A and STAT5B isoforms. AK-2292 induces degradation of STAT5A/B proteins with an outstanding selectivity over all other STAT proteins and more than 6,000 non-STAT proteins, leading to selective inhibition of STAT5 activity in cells. AK-2292 effectively induces STAT5 depletion in normal mouse tissues and human chronic myeloid leukemia (CML) xenograft tissues and achieves tumor regression in two CML xenograft mouse models at well-tolerated dose schedules. AK-2292 is not only a powerful research tool with which to investigate the biology of STAT5 and the therapeutic potential of selective STAT5 protein depletion and inhibition but also a promising lead compound toward ultimate development of a STAT5-targeted therapy.

Discovery of a Potent and Selective STAT5 PROTAC Degrader with Strong Antitumor Activity In Vivo in Acute Myeloid Leukemia.

STAT5 is an attractive therapeutic target for human cancers. We report herein the discovery of a potent and selective STAT5 degrader with strong antitumor activity in vivo. We first obtained small-molecule ligands with sub-micromolar to low micromolar binding affinities to STAT5 and STAT6 SH2 domains and determined co-crystal structures of three such ligands in complex with STAT5A. We successfully transformed these ligands into potent and selective STAT5 degraders using the PROTAC technology with AK-2292 as the best compound. AK-2292 effectively induces degradation of STAT5A, STAT5B, and phosphorylated STAT5 proteins in a concentration- and time-dependent manner in acute myeloid leukemia (AML) cell lines and demonstrates excellent degradation selectivity for STAT5 over all other STAT members. It exerts potent and specific cell growth inhibitory activity in AML cell lines with high levels of phosphorylated STAT5. AK-2292 effectively reduces STAT5 protein in vivo and achieves strong antitumor activity in mice at well-tolerated dose schedules.

Lisaftoclax (APG-2575) Is a Novel BCL-2 Inhibitor with Robust Antitumor Activity in Preclinical Models of Hematologic Malignancy.

PURPOSE: Development of B-cell lymphoma 2 (BCL-2)-specific inhibitors poses unique challenges in drug design because of BCL-2 homology domain 3 (BH3) shared homology between BCL-2 family members and the shallow surface of their protein-protein interactions. We report herein discovery and extensive preclinical investigation of lisaftoclax (APG-2575). EXPERIMENTAL DESIGN: Computational modeling was used to design "lead" compounds. Biochemical binding, mitochondrial BH3 profiling, and cell-based viability or apoptosis assays were used to determine the selectivity and potency of BCL-2 inhibitor lisaftoclax. The antitumor effects of lisaftoclax were also evaluated in several xenograft models. RESULTS: Lisaftoclax selectively binds BCL-2 (Ki < 0.1 nmol/L), disrupts BCL-2:BIM complexes, and compromises mitochondrial outer membrane potential, culminating in BAX/BAK-dependent, caspase-mediated apoptosis. Lisaftoclax exerted strong antitumor activity in hematologic cancer cell lines and tumor cells from patients with chronic lymphocytic leukemia, multiple myeloma, or Waldenström macroglobulinemia. After lisaftoclax treatment, prodeath proteins BCL-2‒like protein 11 (BIM) and Noxa increased, and BIM translocated from cytosol to mitochondria. Consistent with these apoptotic activities, lisaftoclax entered malignant cells rapidly, reached plateau in 2 hours, and significantly downregulated mitochondrial respiratory function and ATP production. Furthermore, lisaftoclax inhibited tumor growth in xenograft models, correlating with caspase activation, poly (ADP-ribose) polymerase 1 cleavage, and pharmacokinetics of the compound. Lisaftoclax combined with rituximab or bendamustine/rituximab enhanced antitumor activity in vivo. CONCLUSIONS: These findings demonstrate that lisaftoclax is a novel, orally bioavailable BH3 mimetic BCL-2-selective inhibitor with considerable potential for the treatment of certain hematologic malignancies.

Mcl-1 levels critically impact the sensitivities of human colorectal cancer cells to APG-1252-M1, a novel Bcl-2/Bcl-XL dual inhibitor that induces Bax-dependent apoptosis.

New treatment options, such as targeted therapies, are urgently needed for the treatment of colorectal cancer (CRC), the third leading cause of cancer-related deaths worldwide. The current study focuses on demonstrating the therapeutic efficacies of APG-1252-M1 (an active form of the prodrug, APG-1252 or pelcitoclax), a highly potent Bcl-2/Bcl-XL dual inhibitor in clinical trials, against CRC and understanding the underlying mechanisms. APG-1252-M1 effectively decreased the survival of CRC cell lines, particularly those expressing relatively low levels of Mcl-1, with the induction of apoptosis. High levels of Mcl-1 were significantly correlated with decreased sensitivity of CRC cell lines to APG-1252-M1. When combined with an Mcl-1 inhibitor, APG-1252-M1 synergistically decreased the survival and induced apoptosis of APG-1252-M1-insensitive cell lines with high levels of Mcl-1. This combination further decreased the survival and enhanced apoptosis even in sensitive cell lines with relatively low levels of Mcl-1, whereas enforced expression of ectopic Mcl-1 in these cells abrogated APG-1252-M1's effects on decreasing cell survival and inducing apoptosis, which could be reversed by Mcl-1 inhibition. APG-1252-M1 rapidly induced cytochrome C and Smac release from mitochondria with caspase-3 and PARP cleavage. Deficiency of Bax in CRC cells abolished APG-1252-M1's ability to induce apoptosis, indicating that APG-1252-M1 induces Bax-dependent apoptosis. The current study thus demonstrates the potential of APG-1252-M1 as a monotherapy in the treatment of CRC, particularly those with low Mcl-1 expression, or in combination with an Mcl-1 inhibitor, warranting further evaluation in vivo and in the clinic.

Design, Synthesis, and Biological Evaluation of Apcin-Based CDC20 Inhibitors.

CDC20 binds to anaphase-promoting complex/cyclosome E3 ubiquitin ligase to recruit substrates for ubiquitination to promote mitotic progression. In breast and other cancers, CDC20 overexpression causes cell cycle dysregulation and is associated with poor prognosis. Apcin was previously discovered as a CDC20 inhibitor exhibiting high micromolar activities. Here, we designed and developed new apcin-based inhibitors by eliminating a controlled substance, chloral hydrate, required for synthesis. We further improved the antitumor activities of the inhibitors by replacing the pyrimidine group with substituted thiazole-containing groups. When evaluated in MDA-MB-231 and MDA-MB-468 triple negative breast cancer cell lines, several analogs showed 5-10-fold improvement over apcin with IC50 values at ∼10 µM in cell viability assays. Tubulin polymerization assay showed our CDC20 inhibitors had no off-target effects against tubulin. Proapoptotic Bim accumulation was detected in our CDC20 inhibitor treated MDA-MB-468 cells. The most effective inhibitors, 22, warrant further development to target CDC20 in diseases.

SD-91 as A Potent and Selective STAT3 Degrader Capable of Achieving Complete and Long-Lasting Tumor Regression.

Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. We report herein our extensive in vitro and in vivo evaluations of SD-91, the product of the hydrolysis of our previously reported STAT3 degrader SD-36. SD-91 binds to STAT3 protein with a high affinity and displays >300-fold selectivity over other STAT family protein members. SD-91 potently and effectively induces degradation of STAT3 protein and displays a high selectivity over other STAT members and >7000 non-STAT proteins in cells. A single administration of SD-91 selectively depletes STAT3 protein in tumor tissues with a persistent effect. SD-91 achieves complete and long-lasting tumor regression in the MOLM-16 xenograft model in mice even with weekly administration. Hence, SD-91 is a potent, highly selective, and efficacious STAT3 degrader for extensive evaluations for the treatment of human cancers and other diseases for which STAT3 plays a key role.

Topography of transcriptionally active chromatin in glioblastoma.

Molecular profiling of the most aggressive brain tumor glioblastoma (GBM) on the basis of gene expression, DNA methylation, and genomic variations advances both cancer research and clinical diagnosis. The enhancer architectures and regulatory circuitries governing tumor-intrinsic transcriptional diversity and subtype identity are still elusive. Here, by mapping H3K27ac deposition, we analyze the active regulatory landscapes across 95 GBM biopsies, 12 normal brain tissues, and 38 cell line counterparts. Analyses of differentially regulated enhancers and super-enhancers uncovered previously unrecognized layers of intertumor heterogeneity. Integrative analysis of variant enhancer loci and transcriptome identified topographies of transcriptional enhancers and core regulatory circuitries in four molecular subtypes of primary tumors: AC1-mesenchymal, AC1-classical, AC2-proneural, and AC3-proneural. Moreover, this study reveals core oncogenic dependency on super-enhancer-driven transcriptional factors, long noncoding RNAs, and druggable targets in GBM. Through profiling of transcriptional enhancers, we provide clinically relevant insights into molecular classification, pathogenesis, and therapeutic intervention of GBM.

Discovery of CJ-2360 as a Potent and Orally Active Inhibitor of Anaplastic Lymphoma Kinase Capable of Achieving Complete Tumor Regression.

We report herein the discovery of a class of potent small-molecule inhibitors of anaplastic lymphoma kinase (ALK) containing a fused indoloquinoline scaffold. The most promising compound CJ-2360 has an IC50 value of 2.2 nM against wild-type ALK and low-nanomolar potency against several clinically reported ALK mutants. This compound is capable of achieving complete tumor regression in the ALK-positive KARPAS-299 xenograft model with oral administration in mice. CJ-2360 represents a promising ALK inhibitor for advanced preclinical development.

Structure-Based Discovery of SD-36 as a Potent, Selective, and Efficacious PROTAC Degrader of STAT3 Protein.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor and an attractive therapeutic target for cancer and other human diseases. Despite 20 years of persistent research efforts, targeting STAT3 has been very challenging. We report herein the structure-based discovery of potent small-molecule STAT3 degraders based upon the proteolysis targeting chimera (PROTAC) concept. We first designed SI-109 as a potent, small-molecule inhibitor of the STAT3 SH2 domain. Employing ligands for cereblon/cullin 4A E3 ligase and SI-109, we obtained a series of potent PROTAC STAT3 degraders, exemplified by SD-36. SD-36 induces rapid STAT3 degradation at low nanomolar concentrations in cells and fails to degrade other STAT proteins. SD-36 achieves nanomolar cell growth inhibitory activity in leukemia and lymphoma cell lines with high levels of phosphorylated STAT3. A single dose of SD-36 results in complete STAT3 protein degradation in xenograft tumor tissue and normal mouse tissues. SD-36 achieves complete and long-lasting tumor regression in the Molm-16 xenograft tumor model at well-tolerated dose-schedules. SD-36 is a potent, selective, and efficacious STAT3 degrader.

A Potent and Selective Small-Molecule Degrader of STAT3 Achieves Complete Tumor Regression In Vivo.

Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. Here we report the discovery of SD-36, a small-molecule degrader of STAT3. SD-36 potently induces the degradation of STAT3 protein in vitro and in vivo and demonstrates high selectivity over other STAT members. Induced degradation of STAT3 results in a strong suppression of its transcription network in leukemia and lymphoma cells. SD-36 inhibits the growth of a subset of acute myeloid leukemia and anaplastic large-cell lymphoma cell lines by inducing cell-cycle arrest and/or apoptosis. SD-36 achieves complete and long-lasting tumor regression in multiple xenograft mouse models at well-tolerated dose schedules. Degradation of STAT3 protein, therefore, is a promising cancer therapeutic strategy.

Small-molecule PROTAC degraders of the Bromodomain and Extra Terminal (BET) proteins - A review.

The PROteolysis TArgeting Chimeric (PROTAC) concept has provided an opportunity for the discovery and development of a completely new type of therapy involving induction of protein degradation. The BET proteins, comprised of BRD2, BRD3, BRD4 and the testis-specific BRDT protein, are epigenetic readers and master transcription coactivators. Extremely potent and efficacious small-molecule PROTAC degraders of the BET proteins, based on available, potent and selective BET inhibitors, have been reported. BET degraders differ from BET inhibitors in their cellular potency, phenotypic effects, pharmacokinetic properties and toxicity profiles. Herein, we provide a review of BET degraders and the differential outcome observed in the cellular and animal models for BET degraders in comparison to BET inhibitors.

MCL-1 inhibition in cancer treatment.

Myeloid cell leukemia-1 (MCL-1), a member of antiapoptotic BCL-2 family proteins, is a key regulator of mitochondrial homeostasis. Frequent overexpression of MCL-1 in human primary and drug-resistant cancer cells makes it an attractive cancer therapeutic target. Significant progress has been made in the development of small-molecule MCL-1 inhibitors in recent years, and three MCL-1 selective inhibitors have advanced to clinical trials. This review briefly discusses recent advances in the development of small molecules targeting MCL-1 for cancer therapy.

Discovery of QCA570 as an Exceptionally Potent and Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of the Bromodomain and Extra-Terminal (BET) Proteins Capable of Inducing Complete and Durable Tumor Regression.

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.