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.

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.

Discovery of M-808 as a Highly Potent, Covalent, Small-Molecule Inhibitor of the Menin-MLL Interaction with Strong In Vivo Antitumor Activity.

Targeting the menin-MLL protein-protein interaction is a new therapeutic strategy for the treatment of acute leukemia carrying MLL fusion (MLL leukemia). We describe herein the structure-based optimization of a class of covalent menin inhibitors, which led to the discovery of M-808 (16) as a highly potent and efficacious covalent menin inhibitor. M-808 effectively inhibits leukemia cell growth at low nanomolar concentrations and is capable of achieving partial tumor regression in an MV4;11 xenograft tumor model in mice at a well-tolerated dose schedule. Determination of the co-crystal structure of M-808 in complex with menin provides a structural basis for their high-affinity, covalent interactions. M-808 represents a promising, covalent menin inhibitor for further optimization and evaluation toward developing a new therapy for the treatment of MLL leukemia.

Discovery of MD-224 as a First-in-Class, Highly Potent, and Efficacious Proteolysis Targeting Chimera Murine Double Minute 2 Degrader Capable of Achieving Complete and Durable Tumor Regression.

Human murine double minute 2 (MDM2) protein is a primary endogenous cellular inhibitor of the tumor suppressor p53 and has been pursued as an attractive cancer therapeutic target. Several potent, nonpeptide, small-molecule inhibitors of MDM2 are currently in clinical development. In this paper, we report our design, synthesis, and evaluation of small-molecule MDM2 degraders based on the proteolysis targeting chimera (PROTAC) concept. The most promising compound (MD-224) effectively induces rapid degradation of MDM2 at concentrations <1 nM in human leukemia cells. It achieves an IC50 value of 1.5 nM in inhibition of growth of RS4;11 cells and also low nanomolar IC50 values in a panel of leukemia cell lines. MD-224 achieves complete and durable tumor regression in vivo in the RS4;11 xenograft tumor model in mice at well-tolerated dose schedules. MD-224 is thus a highly potent and efficacious MDM2 degrader and warrants extensive evaluations as a new class of anticancer agent.

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.

Structure-Based Discovery of CF53 as a Potent and Orally Bioavailable Bromodomain and Extra-Terminal (BET) Bromodomain Inhibitor.

We report the structure-based discovery of CF53 (28) as a highly potent and orally active inhibitor of bromodomain and extra-terminal (BET) proteins. By the incorporation of a NH-pyrazole group into the 9H-pyrimido[4,5- b]indole core, we identified a series of compounds that bind to BRD4 BD1 protein with Ki values of <1 nM and achieve low nanomolar potencies in the cell growth inhibition of leukemia and breast cancer cells. The most-promising compound, CF53, possesses excellent oral pharmacokinetic properties and achieves significant antitumor activity in both triple-negative breast cancer and acute leukemia xenograft models in mice. Determination of the co-crystal structure of CF53 with the BRD4 BD1 protein provides a structural basis for its high binding affinity to BET proteins. CF53 is very selective over non-BET bromodomain-containing proteins. These data establish CF53 as a potent, selective, and orally active BET inhibitor, which warrants further evaluation for advanced preclinical development.

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 a Small-Molecule Degrader of Bromodomain and Extra-Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable of Achieving Tumor Regression.

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.

Discovery of 4-((3'R,4'S,5'R)-6″-Chloro-4'-(3-chloro-2-fluorophenyl)-1'-ethyl-2″-oxodispiro[cyclohexane-1,2'-pyrrolidine-3',3″-indoline]-5'-carboxamido)bicyclo[2.2.2]octane-1-carboxylic Acid (AA-115/APG-115): A Potent and Orally Active Murine Double Minute 2 (MDM2) Inhibitor in Clinical Development.

We previously reported the design of spirooxindoles with two identical substituents at the carbon-2 of the pyrrolidine core as potent MDM2 inhibitors. In this paper we describe an extensive structure-activity relationship study of this class of MDM2 inhibitors, which led to the discovery of 60 (AA-115/APG-115). Compound 60 has a very high affinity to MDM2 (Ki < 1 nM), potent cellular activity, and an excellent oral pharmacokinetic profile. Compound 60 is capable of achieving complete and long-lasting tumor regression in vivo and is currently in phase I clinical trials for cancer treatment.

Targeted Degradation of BET Proteins in Triple-Negative Breast Cancer.

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 protein degrader, BETd-246, which exhibits superior selectivity, potency, and antitumor activity. In human TNBC cells, BETd-246 induced degradation of BET proteins at low nanomolar concentrations within 1 hour 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 with BETi-211 treatment that upregulated and downregulated a similar number of genes. Functional investigations identified the MCL1 gene as a critical downstream effector for 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 that targeting BET proteins for degradation represents an effective therapeutic strategy for TNBC treatment. Cancer Res; 77(9); 2476-87. ©2017 AACR.

Transcellular delivery of vesicular SOCS proteins from macrophages to epithelial cells blunts inflammatory signaling.

JAK-STAT signaling mediates the actions of numerous cytokines and growth factors, and its endogenous brake is the family of SOCS proteins. Consistent with their intracellular roles, SOCS proteins have never been identified in the extracellular space. Here we report that alveolar macrophages can secrete SOCS1 and -3 in exosomes and microparticles, respectively, for uptake by alveolar epithelial cells and subsequent inhibition of STAT activation. Secretion is tunable and occurs both in vitro and in vivo. SOCS secretion into lung lining fluid was diminished by cigarette smoking in humans and mice. Secretion and transcellular delivery of vesicular SOCS proteins thus represent a new model for the control of inflammatory signaling, which is subject to dysregulation during states of inflammation.

Resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in murine models of asthma.

The role and origin of alveolar macrophages (AMs) in asthma are incompletely defined. We sought to clarify these issues in the context of acute allergic lung inflammation using house dust mite and OVA murine models. Use of liposomal clodronate to deplete resident AMs (rAMs) resulted in increased levels of inflammatory cytokines and eosinophil numbers in lavage fluid and augmented the histopathologic evidence of lung inflammation, suggesting a suppressive role for rAMs. Lung digests of asthmatic mice revealed an increased percentage of Ly6C(high)/CD11b(pos) inflammatory monocytes. Clodronate depletion of circulating monocytes, by contrast, resulted in an attenuation of allergic inflammation. A CD45.1/CD45.2 chimera model demonstrated that recruitment at least partially contributes to the AM pool in irradiated nonasthmatic mice, but its contribution was no greater in asthma. Ki-67 staining of AMs supported a role for local proliferation, which was increased in asthma. Our data demonstrate that rAMs dampen, whereas circulating monocytes promote, early events in allergic lung inflammation. Moreover, maintenance of the AM pool in the early stages of asthmatic inflammation depends on local proliferation, but not recruitment.

Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras.

The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38alpha. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.

2-Alkylamino- and alkoxy-substituted 2-amino-1,3,4-oxadiazoles-O-Alkyl benzohydroxamate esters replacements retain the desired inhibition and selectivity against MEK (MAP ERK kinase).

This paper reports a second generation MEK inhibitor. The previously reported potent and efficacious MEK inhibitor, PD-184352 (CI-1040), contains an integral hydroxamate moiety. This compound suffered from less than ideal solubility and metabolic stability. An oxadiazole moiety behaves as a bioisostere for the hydroxamate group, leading to a more metabolically stable and efficacious MEK inhibitor.

The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901.

A novel series of benzhydroxamate esters derived from their precursor anthranilic acids have been prepared and have been identified as potent MEK inhibitors. 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide, CI-1040, was the first MEK inhibitor to demonstrate in vivo activity in preclinical animal models and subsequently became the first MEK inhibitor to enter clinical trial. CI-1040 suffered however from poor exposure due to its poor solubility and rapid clearance, and as a result, development of the compound was terminated. Optimization of the diphenylamine core and modification of the hydroxamate side chain for cell potency, solubility, and exposure with oral delivery resulted in the discovery of the clinical candidate N-(2,3-dihydroxy-propoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide PD 0325901.

N-(6,7-dichloro-2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-N-alkylsulfonamides as peripherally restricted N-methyl-D-aspartate receptor antagonists for the treatment of pain.

It has been hypothesized that peripherally restricted NMDA receptor antagonists may be effective analgesics for osteoarthritis pain. A class of novel quinoxalinedione atropisomers, first discovered for an NMDA receptor antagonist program for the treatment of stroke, was evaluated and further optimized with the goal of finding peripherally restricted NMDA receptor antagonists.

A role for K-ras in conferring resistance to the MEK inhibitor, CI-1040.

PD184352/CI-1040 is a potent and selective MEK1/2 inhibitor that represents the first MEK-targeted agent to enter clinical trials. Here, we report the development and molecular characterization of CI-1040 resistance in the murine colon 26 (C26) carcinoma cell line. The growth rate of the resistant line (C26/CI-1040r) in the presence of 2 microM CI-1040 is comparable to that of parental C26 cells in the absence of CI-1040. C26/CI-1040r cells are approximately 100-fold more resistant than the parental line to CI-1040 inhibition in soft agar and are less sensitive to the induction of apoptosis that normally occurs in response to CI-1040 treatment. K-ras expression is significantly elevated in C26/CI-1040r cells. We confirmed a causative role for K-ras in conferring resistance to CI-1040 by transfecting K-ras into parental C26 cells, whereupon an elevation in the levels of phosphorylated ERK1/2 was observed in addition to resistance to CI-1040. Furthermore, an in vivo-derived MEK inhibitor-resistant line also shows increased K-ras expression. Our data suggest that increasing activated K-ras expression represents one potential mechanism by which tumor cells that initially are responsive to blockade of the MAP kinase pathway can overcome their sensitivity to MEK inhibition.