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 EC
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.
The three-dimensional conformations adopted by a free ligand in solution impact bioactivity and physicochemical properties. Solution 1D NMR spectra inherently contain information on ligand ...conformational flexibility and three-dimensional shape, as well as the propensity of the free ligand to fully preorganize into the bioactive conformation. Herein we discuss some key learnings, distilled from our experience developing potent and selective synthetic macrocyclic inhibitors, including Mcl-1 clinical candidate AZD5991. Case studies have been selected from recent oncology research projects, demonstrating how 1D NMR conformational signatures can complement X-ray protein–ligand structural information to guide medicinal chemistry optimization. Learning to extract free ligand conformational information from routinely available 1D NMR signatures has proven to be fast enough to guide medicinal chemistry decisions within design cycles for compound optimization.
B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report ...potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.
ATM inhibitors, such as 7, have demonstrated the antitumor potential of ATM inhibition when combined with DNA double-strand break-inducing agents in mouse xenograft models. However, the properties of ...7 result in a relatively high predicted clinically efficacious dose. In an attempt to minimize attrition during clinical development, we sought to identify ATM inhibitors with a low predicted clinical dose (<50 mg) and focused on strategies to increase both ATM potency and predicted human pharmacokinetic half-life (predominantly through the increase of volume of distribution). These efforts resulted in the discovery of 64 (AZD0156), an exceptionally potent and selective inhibitor of ATM based on an imidazo4,5-cquinolin-2-one core. 64 has good preclinical phamacokinetics, a low predicted clinical dose, and a high maximum absorbable dose. 64 has been shown to potentiate the efficacy of the approved drugs irinotecan and olaparib in disease relevant mouse models and is currently undergoing clinical evaluation with these agents.
The first large scale analysis of in vitro absorption, distribution, metabolism, excretion, and toxicity (ADMET) data shared across multiple major pharma has been performed. Using advanced matched ...molecular pair analysis (MMPA), we combined data from three pharmaceutical companies and generated ADMET rules, avoiding the need to disclose the full chemical structures. On top of the very large exchange of knowledge, all companies involved synergistically gained approximately 20% more rules from the shared transformations. There is good quantitative agreement between the rules based on shared data compared to both individual companies' rules and rules published in the literature. Known correlations between log D, solubility, in vitro clearance, and plasma protein binding also hold in transformation space, but there are also interesting exceptions. Data pools such as this allow focusing on particular functional groups and characterizing their ADMET profile. Finally the role of a corpus of robustly tested medicinal chemistry knowledge in the training of medicinal chemistry is discussed.
Inhibition of the protein–protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and ...profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo1,5-apyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand–protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.
The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by ...relative binding free energy calculations, resulted in AZ’9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.
The inhibition of ataxia-telangiectasia mutated (ATM) has been shown to chemo- and radio-sensitize human glioma cells in vitro and therefore might provide an exciting new paradigm in the treatment of ...glioblastoma multiforme (GBM). The effective treatment of GBM will likely require a compound with the potential to efficiently cross the blood–brain barrier (BBB). Starting from clinical candidate AZD0156, 4, we investigated the imidazoquinolin-2-one scaffold with the goal of improving likely CNS exposure in humans. Strategies aimed at reducing hydrogen bonding, basicity, and flexibility of the molecule were explored alongside modulating lipophilicity. These studies identified compound 24 (AZD1390) as an exceptionally potent and selective inhibitor of ATM with a good preclinical pharmacokinetic profile. 24 showed an absence of human transporter efflux in MDCKII-MDR1-BCRP studies (efflux ratio <2), significant BBB penetrance in nonhuman primate PET studies (K p,uu 0.33) and was deemed suitable for development as a clinical candidate to explore the radiosensitizing effects of ATM in intracranial malignancies.
Casitas B-lymphoma proto-oncogene-b (Cbl-b), a member of the Cbl family of RING finger E3 ubiquitin ligases, has been demonstrated to play a central role in regulating effector T-cell function. ...Multiple studies using gene-targeting approaches have provided direct evidence that Cbl-b negatively regulates T, B, and NK cell activation via a ubiquitin-mediated protein modulation. Thus, inhibition of Cbl-b ligase activity can lead to immune activation and has therapeutic potential in immuno-oncology. Herein, we describe the discovery and optimization of an arylpyridone series as Cbl-b inhibitors by structure-based drug discovery to afford compound 31. This compound binds to Cbl-b with an IC50 value of 30 nM and induces IL-2 production in T-cells with an EC50 value of 230 nM. Compound 31 also shows robust intracellular target engagement demonstrated through inhibition of Cbl-b autoubiquitination, inhibition of ubiquitin transfer to ZAP70, and the cellular modulation of phosphorylation of a downstream signal within the TCR axis.
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