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.

Compliance With the National SEP-1 Quality Measure and Association With Sepsis Outcomes: A Multicenter Retrospective Cohort Study.

OBJECTIVES: Many septic patients receive care that fails the Centers for Medicare and Medicaid Services' SEP-1 measure, but it is unclear whether this reflects meaningful lapses in care, differences in clinical characteristics, or excessive rigidity of the "all-or-nothing" measure. We compared outcomes in cases that passed versus failed SEP-1 during the first 2 years after the measure was implemented. DESIGN: Retrospective cohort study. SETTING: Seven U.S. hospitals. PATIENTS: Adult patients included in SEP-1 reporting between October 2015 and September 2017. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of 851 sepsis cases in the cohort, 281 (33%) passed SEP-1 and 570 (67%) failed. SEP-1 failures had higher rates of septic shock (20% vs 9%; p < 0.001), hospital-onset sepsis (11% vs 4%; p = 0.001), and vague presenting symptoms (46% vs 30%; p < 0.001). The most common reasons for failure were omission of 3- and 6-hour lactate measurements (228/570 failures, 40%). Only 86 of 570 failures (15.1%) had greater than 3-hour delays until broad-spectrum antibiotics. Cases that failed SEP-1 had higher in-hospital mortality rates (18.4% vs 11.0%; odds ratio, 1.82; 95% CI, 1.19-2.80; p = 0.006), but this association was no longer significant after adjusting for differences in clinical characteristics and severity of illness (adjusted odds ratio, 1.36; 95% CI, 0.85-2.18; p = 0.205). Delays of greater than 3 hours until antibiotics were significantly associated with death (adjusted odds ratio, 1.94; 95% CI, 1.04-3.62; p = 0.038), whereas failing SEP-1 for any other reason was not (adjusted odds ratio, 1.10; 95% CI, 0.70-1.72; p = 0.674). CONCLUSIONS: Crude mortality rates were higher in sepsis cases that failed versus passed SEP-1, but there was no difference after adjusting for clinical characteristics and severity of illness. Delays in antibiotic administration were associated with higher mortality but only accounted for a small fraction of SEP-1 failures. SEP-1 may not clearly differentiate between high- and low-quality care, and detailed risk adjustment is necessary to properly interpret associations between SEP-1 compliance and mortality.

Design of the First-in-Class, Highly Potent Irreversible Inhibitor Targeting the Menin-MLL Protein-Protein Interaction.

The structure-based design of M-525 as the first-in-class, highly potent, irreversible small-molecule inhibitor of the menin-MLL interaction is presented. M-525 targets cellular menin protein at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL-regulated gene expression in MLL leukemia cells. M-525 demonstrates high cellular specificity over non-MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co-crystal structure of M-525 in complex with menin firmly establish its mode of action. A single administration of M-525 effectively suppresses MLL-regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M-525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.

Discovery of MD-265: A Potent MDM2 Degrader That Achieves Complete Tumor Regression and Improves Long-Term Survival of Mice with Leukemia.

MDM2 has been pursued as an attractive therapeutic target for human cancers. Herein, we describe our discovery of MD-265 as a promising PROTAC MDM2 degrader and extensive in vitro and in vivo evaluations of its therapeutic potential and mechanism of action. MD-265 effectively depleted MDM2 protein in cancer cells at concentrations as low as 1 nM, leading to strong activation of p53 in cancer cells carrying wild-type p53. It selectively inhibited the growth of wild-type p53 leukemia cell lines and showed no activity in mutated p53 lines. MD-265 achieved persistent tumor regression in a leukemia xenograft model without causing any signs of toxicity and dramatically improved survival of mice in a disseminated leukemia model even with a weekly administration. MD-265 displayed an excellent intravenous PK profile in mice, rats, and dogs. MD-265 is a promising MDM2 degrader for advanced preclinical development for the treatment 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 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.

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 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 ERD-1233 as a Potent and Orally Efficacious Estrogen Receptor PROTAC Degrader for the Treatment of ER+ Human Breast Cancer.

Despite the development of highly effective therapies for the treatment of estrogen receptor α (ERα)-positive human breast cancer, clinical resistance to current therapies requires the development of novel therapeutic strategies. Herein, we report the discovery of ERD-1233 as a potent and orally efficacious ERα degrader designed using the PROTAC technology. ERD-1233 was developed based on Lasofoxifene as the ER binding moiety and a novel cereblon ligand through extensive optimization of the linker. ERD-1233 potently and effectively reduces the ERα protein in vitro and achieves excellent oral bioavailability in mice and rats. Oral administration of ERD-1233 effectively reduces ER protein in ER+ tumors and achieves tumor regression in the ER wild-type MCF-7 xenograft tumor model and strong tumor growth inhibition in the ESR1Y537S mutated model in mice. Our data demonstrate that ERD-1233 is a promising ER PROTAC degrader for extensive evaluation as a new therapy for the treatment of ER+ human breast cancer.

Diastereomeric spirooxindoles as highly potent and efficacious MDM2 inhibitors.

Small-molecule inhibitors that block the MDM2-p53 protein-protein interaction (MDM2 inhibitors) are being intensely pursued as a new therapeutic strategy for cancer treatment. We previously published a series of spirooxindole-containing compounds as a new class of MDM2 small-molecule inhibitors. We report herein a reversible ring-opening-cyclization reaction for some of these spirooxindoles, which affords four diastereomers from a single compound. Our biochemical binding data showed that the stereochemistry in this class of compounds has a major effect on their binding affinities to MDM2, with >100-fold difference between the most potent and the least potent stereoisomers. Our study has led to the identification of a set of highly potent MDM2 inhibitors with a stereochemistry that is different from that of our previously reported compounds. The most potent compound (MI-888) binds to MDM2 with a Ki value of 0.44 nM and achieves complete and long-lasting tumor regression in an animal model of human cancer.

Physiologically based pharmacokinetic and pharmacodynamic modeling of an antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in a mouse xenograft model of human breast cancer.

The inhibitors of apoptosis proteins (IAPs) are a class of key apoptosis regulators overexpressed or dysregulated in cancer. SM-406/AT-406 is a potent and selective small molecule mimetic of Smac that antagonizes the inhibitor of apoptosis proteins (IAPs). A physiologically based pharmacokinetic and pharmacodynamic (PBPK-PD) model was developed to predict the tissue concentration-time profiles of SM-406, the related onco-protein levels in tumor, and the tumor growth inhibition in a mouse model bearing human breast cancer xenograft. In the whole body physiologically based pharmacokinetic (PBPK) model for pharmacokinetics characterization, a well stirred (perfusion rate-limited) model was used to describe SM-406 pharmacokinetics in the lung, heart, kidney, intestine, liver and spleen, and a diffusion rate-limited (permeability limited) model was used for tumor. Pharmacodynamic (PD) models were developed to correlate the SM-406 concentration in tumor to the cIAP1 degradation, pro-caspase 8 decrease, CL-PARP accumulation and tumor growth inhibition. The PBPK-PD model well described the experimental pharmacokinetic data, the pharmacodynamic biomarker responses and tumor growth. This model may be helpful to predict tumor and plasma SM-406 concentrations in the clinic.

Precise Conformational Control Yielding Highly Potent and Exceptionally Selective BRD4 Degraders with Strong Antitumor Activity.

Starting from a nonselective bromodomain and extraterminal (BET) inhibitor and a cereblon ligand, we have used precise conformational control for the development of two potent and highly selective BRD4 degraders, BD-7148 and BD-9136. These compounds induce rapid degradation of BRD4 protein in cells at concentrations as low as 1 nM and demonstrate ≥1000-fold degradation selectivity over BRD2 or BRD3 protein. Proteomic analysis of >5700 proteins confirmed their highly selective BRD4 degradation. A single dose of BD-9136 selectively and effectively depletes BRD4 protein in tumor tissues for >48 h. BD-9136 effectively inhibits tumor growth without adverse effects on mice and is more efficacious than the corresponding pan BET inhibitor. This study suggests selective degradation of BRD4 as a strategy for the treatment of human cancers and demonstrates a strategy for the design of highly selective PROTAC degraders.

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.

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.

Discovery of ARD-2051 as a Potent and Orally Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor for the Treatment of Advanced Prostate Cancer.

We report the discovery of ARD-2051 as a potent and orally efficacious androgen receptor (AR) proteolysis-targeting chimera degrader. ARD-2051 achieves DC50 values of 0.6 nM and Dmax >90% in inducing AR protein degradation in both the LNCaP and VCaP prostate cancer cell lines, potently and effectively suppresses AR-regulated genes, and inhibits cancer cell growth. ARD-2051 achieves a good oral bioavailability and pharmacokinetic profile in mouse, rat, and dog. A single oral dose of ARD-2051 strongly reduces AR protein and suppresses AR-regulated gene expression in the VCaP xenograft tumor tissue in mice. Oral administration of ARD-2051 effectively inhibits VCaP tumor growth and causes no signs of toxicity in mice. ARD-2051 is a promising AR degrader for advanced preclinical development for the treatment of AR+ human cancers.

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.

Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition.

We have designed MI-219 as a potent, highly selective and orally active small-molecule inhibitor of the MDM2-p53 interaction. MI-219 binds to human MDM2 with a K(i) value of 5 nM and is 10,000-fold selective for MDM2 over MDMX. It disrupts the MDM2-p53 interaction and activates the p53 pathway in cells with wild-type p53, which leads to induction of cell cycle arrest in all cells and selective apoptosis in tumor cells. MI-219 stimulates rapid but transient p53 activation in established tumor xenograft tissues, resulting in inhibition of cell proliferation, induction of apoptosis, and complete tumor growth inhibition. MI-219 activates p53 in normal tissues with minimal p53 accumulation and is not toxic to animals. MI-219 warrants clinical investigation as a new agent for cancer treatment.

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.

Selective inhibition of cullin 3 neddylation through covalent targeting DCN1 protects mice from acetaminophen-induced liver toxicity.

Cullin-RING E3 ligases (CRLs) regulate the turnover of approximately 20% of mammalian cellular proteins. Neddylation of individual cullin proteins is essential for the activation of each CRL. We report herein the discovery of DI-1548 and DI-1859 as two potent, selective and covalent DCN1 inhibitors. These inhibitors selectively inhibit neddylation of cullin 3 in cells at low nanomolar concentrations and are 2-3 orders of magnitude more potent than our previously reported reversible DCN1 inhibitor. Mass spectrometric analysis and co-crystal structures reveal that these compounds employ a unique mechanism of covalent bond formation with DCN1. DI-1859 induces a robust increase of NRF2 protein, a CRL3 substrate, in mouse liver and effectively protects mice from acetaminophen-induced liver damage. Taken together, this study demonstrates the therapeutic potential of selective inhibition of cullin neddylation.