Optimization of a series of novel, potent and selective Macrocyclic SYK inhibitors.

Spleen tyrosine kinase (SYK) is a non-receptor cytoplasmic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signalling, inhibition of SYK has been a target of interest in a variety of diseases. Herein, we report the use of structure-based drug design to discover a series of potent macrocyclic inhibitors of SYK, with excellent kinome selectivity and in vitro metabolic stability. We were able to remove hERG inhibition through the optimization of physical properties, and utilized a pro-drug strategy to address permeability challenges.

Targeting Bfl-1 via acute CDK9 inhibition overcomes intrinsic BH3-mimetic resistance in lymphomas.

BH3 mimetics like venetoclax target prosurvival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. We demonstrate that endogenous Bfl-1 expression can render preclinical lymphoma tumor models insensitive to Mcl-1 and Bcl-2 inhibitors. However, suppression of Bfl-1 alone was insufficient to fully induce apoptosis in Bfl-1-expressing lymphomas, highlighting the need for targeting additional prosurvival proteins in this context. Importantly, we demonstrated that cyclin-dependent kinase 9 (CDK9) inhibitors rapidly downregulate both Bfl-1 and Mcl-1, inducing apoptosis in BH3-mimetic-resistant lymphoma cell lines in vitro and driving in vivo tumor regressions in diffuse large B-cell lymphoma patient-derived xenograft models expressing Bfl-1. These data underscore the need to clinically develop CDK9 inhibitors, like AZD4573, for the treatment of lymphomas using Bfl-1 as a selection biomarker.

Novel potent and selective pyrazolylpyrimidine-based SYK inhibitors.

Hybridisation of amino-pyrimidine based SYK inhibitors (e.g. 1a) with previously reported diamine-based SYK inhibitors (e.g. TAK-659) led to the identification and optimisation of a novel pyrimidine-based series of potent and selective SYK inhibitors, where the original aminomethylene group was replaced by a 3,4-diaminotetrahydropyran group. The initial compound 5 achieved excellent SYK potency. However, it suffered from poor permeability and modest kinase selectivity. Further modifications of the 3,4-diaminotetrahydropyran group were identified and the interactions of those groups with Asp512 were characterised by protein X-ray crystallography. Further optimisation of this series saw mixed results where permeability and kinase selectivity were increased and oral bioavailability was achieved in the series, but at the expense of potent hERG inhibition.

Identification of a novel series of azabenzimidazole-derived inhibitors of spleen tyrosine kinase.

Spleen Tyrosine Kinase (SYK) is a well-studied enzyme with therapeutic applications in oncology and autoimmune diseases. We identified an azabenzimidazole (ABI) series of SYK inhibitors by mining activity data of 86,000 compounds from legacy biochemical assays with SYK and other homologous kinases as target enzymes. A structure-based design and hybridization approach was then used to improve the potency and kinase selectivity of the hits. Lead compound 23 from this novel ABI series has a SYK IC50 = 0.21 nM in a biochemical assay and inhibits growth of SUDHL-4 cells at a GI50 = 210 nM.

Optimization of a series of potent, selective and orally bioavailable SYK inhibitors.

Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.

AZD4573 Is a Highly Selective CDK9 Inhibitor That Suppresses MCL-1 and Induces Apoptosis in Hematologic Cancer Cells.

PURPOSE: Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and potential therapeutic target for many cancers. Multiple nonselective CDK9 inhibitors have progressed clinically but were limited by a narrow therapeutic window. This work describes a novel, potent, and highly selective CDK9 inhibitor, AZD4573. EXPERIMENTAL DESIGN: The antitumor activity of AZD4573 was determined across broad cancer cell line panels in vitro as well as cell line- and patient-derived xenograft models in vivo. Multiple approaches, including integrated transcriptomic and proteomic analyses, loss-of-function pathway interrogation, and pharmacologic comparisons, were employed to further understand the major mechanism driving AZD4573 activity and to establish an exposure/effect relationship. RESULTS: AZD4573 is a highly selective and potent CDK9 inhibitor. It demonstrated rapid induction of apoptosis and subsequent cell death broadly across hematologic cancer models in vitro, and MCL-1 depletion in a dose- and time-dependent manner was identified as a major mechanism through which AZD4573 induces cell death in tumor cells. This pharmacodynamic (PD) response was also observed in vivo, which led to regressions in both subcutaneous tumor xenografts and disseminated models at tolerated doses both as monotherapy or in combination with venetoclax. This understanding of the mechanism, exposure, and antitumor activity of AZD4573 facilitated development of a robust pharmacokinetic/PD/efficacy model used to inform the clinical trial design. CONCLUSIONS: Selective targeting of CDK9 enables the indirect inhibition of MCL-1, providing a therapeutic option for MCL-1-dependent diseases. Accordingly, AZD4573 is currently being evaluated in a phase I clinical trial for patients with hematologic malignancies (clinicaltrials.gov identifier: NCT03263637).See related commentary by Alcon et al., p. 761.

Potent and selective bivalent inhibitors of BET bromodomains.

Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC50 of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.

AZD5153: A Novel Bivalent BET Bromodomain Inhibitor Highly Active against Hematologic Malignancies.

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563-74. ©2016 AACR.

Fabrication of carbohydrate microarrays on a poly(2-hydroxyethyl methacrylate)-based photoactive substrate.

We report the fabrication of carbohydrate microarrays on a photoactive polymer, poly(HEMA-co-HEMA-PFPA), synthesized by RAFT copolymerization of 2-hydroxyethyl methacrylate (HEMA) and perfluorophenyl azide (PFPA)-derivatized HEMA (HEMA-PFPA). PFPA allows the covalent immobilization of carbohydrates whereas the HEMA polymer provides an antifouling surface, thus the microarrays can be used directly without pretreating the array with a blocking agent. The microarrays were prepared by spin-coating the polymer followed by printing the carbohydrates. Subsequent irradiation simultaneously immobilized the carbohydrates and crosslinked the polymer matrix. The obtained 3D carbohydrate microarrays showed enhanced fluorescence signals upon treating with a fluorescent lectin in comparison with a 2D microarray. The signals were acquired at a lower lectin concentration and a shorter incubation time. When treated with E. coli bacteria, the carbohydrate microarray showed results that were consistent with their binding patterns.