Identification of Novel Potent NSD2-PWWP1 Ligands Using Structure-Based Design and Computational Approaches.

Dysregulation of histone methyl transferase nuclear receptor-binding SET domain 2 (NSD2) has been implicated in several hematological and solid malignancies. NSD2 is a large multidomain protein that carries histone writing and histone reading functions. To date, identifying inhibitors of the enzymatic activity of NSD2 has proven challenging in terms of potency and SET domain selectivity. Inhibition of the NSD2-PWWP1 domain using small molecules has been considered as an alternative approach to reduce NSD2-unregulated activity. In this article, we present novel computational chemistry approaches, encompassing free energy perturbation coupled to machine learning (FEP/ML) models as well as virtual screening (VS) activities, to identify high-affinity NSD2 PWWP1 binders. Through these activities, we have identified the most potent NSD2-PWWP1 binder reported so far in the literature: compound 34 (pIC50 = 8.2). The compounds identified herein represent useful tools for studying the role of PWWP1 domains for inhibition of human NSD2.

Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

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.

Discovery and Optimization of the First ATP Competitive Type-III c-MET Inhibitor.

Recent clinical reports have highlighted the need for wild-type (WT) and mutant dual inhibitors of c-MET kinase for the treatment of cancer. We report herein a novel chemical series of ATP competitive type-III inhibitors of WT and D1228V mutant c-MET. Using a combination of structure-based drug design and computational analyses, ligand 2 was optimized to a highly selective chemical series with nanomolar activities in biochemical and cellular settings. Representatives of the series demonstrate excellent pharmacokinetic profiles in rat in vivo studies with promising free-brain exposures, paving the way for the design of brain permeable drugs for the treatment of c-MET driven cancers.

Discovery of a selective c-MET inhibitor with a novel binding mode.

The c-MET receptor tyrosine kinase has received considerable attention as a cancer drug target yet there remains a need for inhibitors which are selective for c-MET and able to target emerging drug-resistant mutants. We report here the discovery, by screening a DNA-encoded chemical library, of a highly selective c-MET inhibitor which was shown by X-ray crystallography to bind to the kinase in an unprecedented manner. These results represent a novel mode of inhibiting c-MET with a small molecule and may provide a route to targeting drug-resistant forms of the kinase whilst avoiding potential toxicity issues associated with broad kinome inhibition.

Discovery of a Potent and Selective ATAD2 Bromodomain Inhibitor with Antiproliferative Activity in Breast Cancer Models.

ATAD2 is an epigenetic bromodomain-containing target which is overexpressed in many cancers and has been suggested as a potential oncology target. While several small molecule inhibitors have been described in the literature, their cellular activity has proved to be underwhelming. In this work, we describe the identification of a novel series of ATAD2 inhibitors by high throughput screening, confirmation of the bromodomain region as the site of action, and the optimization campaign undertaken to improve the potency, selectivity, and permeability of the initial hit. The result is compound 5 (AZ13824374), a highly potent and selective ATAD2 inhibitor which shows cellular target engagement and antiproliferative activity in a range of breast cancer models.

Phosphorylation-dependent BRD4 dimerization and implications for therapeutic inhibition of BET family proteins.

Bromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains. Phosphorylation by casein kinase II (CK2) regulates BRD4 function, is necessary for active transcription and is involved in resistance to BRD4 drug inhibition in triple-negative breast cancer. Here, we provide the first biophysical analysis of BRD4 phospho-regulation. Using integrative structural biology, we show that phosphorylation by CK2 modulates the dimerization of human BRD4. We identify two conserved regions, a coiled-coil motif and the Basic-residue enriched Interaction Domain (BID), essential for the BRD4 structural rearrangement, which we term the phosphorylation-dependent dimerization domain (PDD). Finally, we demonstrate that bivalent inhibitors induce a conformational change within BRD4 dimers in vitro and in cancer cells. Our results enable the proposal of a model for BRD4 activation critical for the characterization of its protein-protein interaction network and for the development of more specific therapeutics.

Selective CRAF Inhibition Elicits Transactivation.

Discovering molecules that regulate closely related protein isoforms is challenging, and in many cases the consequences of isoform-specific pharmacological regulation remains unknown. RAF isoforms are commonly mutated oncogenes that serve as effector kinases in MAP kinase signaling. BRAF/CRAF heterodimers are believed to be the primary RAF signaling species, and many RAF inhibitors lead to a "paradoxical activation" of RAF kinase activity through transactivation of the CRAF protomer; this leads to resistance mechanisms and secondary tumors. It has been hypothesized that CRAF-selective inhibition might bypass paradoxical activation, but no CRAF-selective inhibitor has been reported and the consequences of pharmacologically inhibiting CRAF have remained unknown. Here, we use bio-orthogonal ligand tethering (BOLT) to selectively target inhibitors to CRAF. Our results suggest that selective CRAF inhibition promotes paradoxical activation and exemplify how BOLT may be used to triage potential targets for drug discovery before any target-selective small molecules are known.

Structural and Molecular Insight into Resistance Mechanisms of First Generation cMET Inhibitors.

Many small molecule inhibitors of the cMET receptor tyrosine kinase have been evaluated in clinical trials for the treatment of cancer and resistance-conferring mutations of cMET are beginning to be reported for a number of such compounds. There is now a need to understand specific cMET mutations at the molecular level, particularly concerning small molecule recognition. Toward this end, we report here the first crystal structures of the recent clinically observed resistance-conferring D1228V cMET mutant in complex with small molecule inhibitors, along with a crystal structure of wild-type cMET bound by the clinical compound savolitinib and supporting cellular, biochemical, and biophysical data. Our findings indicate that the D1228V alteration induces conformational changes in the kinase, which could have implications for small molecule inhibitor design. The data we report here increases our molecular understanding of the D1228V cMET mutation and provides insight for future inhibitor design.

Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1.

Methods to measure cellular target engagement are increasingly being used in early drug discovery. The Cellular Thermal Shift Assay (CETSA) is one such method. CETSA can investigate target engagement by measuring changes in protein thermal stability upon compound binding within the intracellular environment. It can be performed in high-throughput, microplate-based formats to enable broader application to early drug discovery campaigns, though high-throughput forms of CETSA have only been reported for a limited number of targets. CETSA offers the advantage of investigating the target of interest in its physiological environment and native state, but it is not clear yet how well this technology correlates to more established and conventional cellular and biochemical approaches widely used in drug discovery. We report two novel high-throughput CETSA (CETSA HT) assays for B-Raf and PARP1, demonstrating the application of this technology to additional targets. By performing comparative analyses with other assays, we show that CETSA HT correlates well with other screening technologies and can be applied throughout various stages of hit identification and lead optimization. Our results support the use of CETSA HT as a broadly applicable and valuable methodology to help drive drug discovery campaigns to molecules that engage the intended target in cells.

Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics.

The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.

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.

Sustained wash-resistant receptor activation responses of GPR119 agonists.

G protein-coupled receptor 119 (GPR119) is involved in regulating metabolic homoeostasis, with GPR119 agonists targeted for the treatment of type-2 diabetes and obesity. Using the endogenous agonist oleoylethanolamide and a number of small molecule synthetic agonists we have investigated the temporal dynamics of receptor signalling. Using both a dynamic luminescence biosensor-based assay and an endpoint cAMP accumulation assay we show that agonist-driven desensitization is not a major regulatory mechanism for GPR119 despite robust activation responses, regardless of the agonist used. Temporal analysis of the cAMP responses demonstrated sustained signalling resistant to washout for some, but not all of the agonists tested. Further analysis indicated that the sustained effects of one synthetic agonist AR-231,453 were consistent with a role for slow dissociation kinetics. In contrast, the sustained responses to MBX-2982 and AZ1 appeared to involve membrane deposition. We also detect wash-resistant responses to AR-231,453 at the level of physiologically relevant responses in an endogenous expression system (GLP-1 secretion in GLUTag cells). In conclusion, our findings indicate that in a recombinant expression system GPR119 activation is sustained, with little evidence of pronounced receptor desensitization, and for some ligands persistent agonist responses continue despite removal of excess agonist. This provides novel understanding of the temporal responses profiles of potential drug candidates targetting GPR119, and highlights the importance of carefully examining the the mechanisms through which GPCRs generate sustained responses.

An evaluation of fluorescence polarization and lifetime discriminated polarization for high throughput screening of serine/threonine kinases.

We used two kinases, c-jun N terminal kinase (JNK-1) and protein kinase C (PKC), as model enzymes to evaluate the potential of fluorescence polarization (FP) for high-throughput screening and the susceptibility of these assays to compound interference. For JNK-1 the enzyme kinetics in the FP assay were consistent with those found in a [gamma-33P]ATP filter wash assay. Determined pIC(50)s for nonfluorescent JNK-1 inhibitors were also consistent with those found in the filter wash assay. In contrast, fluorescent compounds were found to interfere with the JNK-1 FP assay, appearing as false positives, defined by their lack of activity in the filter wash assay. We also developed a second assay using a different kinase, protein kinase C, which was used to test a 5000 compound diversity set. As for JNK-1, interference from fluorescent compounds caused a high false positive rate. The Molecular Devices Corporation 'FLARe' instrument is capable of discriminating between fluorophores on the basis of their fluorescence (excited state) lifetime, and may assist in reducing compound interference in fluorescent assays. In both model FP kinase assays described here some, although not complete, reduction in interference from fluorescent compounds was achieved by the use of FLARe.

Studies on quinazolinones as dual inhibitors of Pgp and MRP1 in multidrug resistance.

The syntheses and SAR studies of various quinazolinone compounds are described for the dual inhibition of Pgp and MRP1 in multidrug resistance.