Aminopyrazine Inhibitors Binding to an Unusual Inactive Conformation of the Mitotic Kinase Nek2: SAR and Structural Characterization†

We report herein the first systematic exploration of inhibitors of the mitotic kinase Nek2. Starting from HTS hit aminopyrazine 2, compounds with improved activity were identified using structure-based design. Our structural biology investigations reveal two notable observations. First, 2 and related compounds bind to an unusual, inactive conformation of the kinase which to the best of our knowledge has not been reported for other types of kinase inhibitors. Second, a phenylalanine residue at the center of the ATP pocket strongly affects the ability of the inhibitor to bind to the protein. The implications of these observations are discussed, and the work described here defines key features for potent and selective Nek2 inhibition, which will aid the identification of more advanced inhibitors of Nek2

Molecular strategies to reduce unnecessary repeat prostate biopsies of men with elevated serum PSA

Prostate cancer (PCa) is the most common cancer in men. It is a heterogeneous disease and currently there are no reliable biomarkers available to stratify men for prostate biopsy (PBx) and treatment. Hence, there is a risk of over-diagnosing insignificant disease, or under-diagnosing significant disease. We aimed to evaluate FDA approved Prostate Cancer gene 3 (PCA3, FDA approved) and N6-methyladenosine (m6A) for diagnostic properties. PCA3 is a long non-coding RNA (ncRNA) that is unstable, has an unclear biological role and is expensive to chemically treat to prevent degradation prior to analysis. Furthermore, the biological role of this RNA is unclear. Long ncRNAs are degraded into shorter forms, we explored whether this was the fatecase for PCA3. We identified a short segment of RNA within intron 1 of PCA3 bioinformatically which we termed PCA3 short RNA2 (PCA3-shRNA2). The and showed that PCA3-shRNA2 expression of this short RNA correlated to that of PCA3 in PCa cell lines, urinary samples and PBx tissue. PCA3-shRNA2 was overexpressed in urinary samples obtained from men with PCa compared to BPH, was regulated by testosterone and had a diagnostic accuracy similar to that of PCA3. We identified oncogenic mRNA targets of PCA3-shRNA2 and that are involved in oncogenesis and found that COPS2 was underexpressed in cancerous urinary samples. There are over a hundred RNA modifications described and methylation of N6-adenosine base is the most common methylated site. m6A is reversible and may be, involved in oncogenesis. and has recently been mapped throughout the transcriptome. We profiled m6A in PCa cell lines by immunoprecipitation and RNA sequencing, and found interesting oncogenic RNAs (e.g. PARG,) that were differentially expressed in LNCaP-LN3 cells. We identified a novel RNA within PCA3 that is stable, easy to measure, overexpressed in PCa samples and appeared to target oncogenic mRNAs. We profiled m6A in PCa cell lines and have identified important N6-adenosine methylated RNAs associated with PCa development. In conclusion PCA3-shRNA2 and m6A have evolving roles in cancer and may function well as biomarkers

Novel Quinazolinone Inhibitors of ALK2 Flip between Alternate Binding Modes: Structure–Activity Relationship, Structural Characterization, Kinase Profiling, and Cellular Proof of Concept

Structure–activity relationship and crystallographic data revealed that quinazolinone-containing fragments flip between two distinct modes of binding to activin receptor-like kinase-2 (ALK2). We explored both binding modes to discover potent inhibitors and characterized the chemical modifications that triggered the flip in binding mode. We report kinase selectivity and demonstrate that compounds of this series modulate ALK2 in cancer cells. These inhibitors are attractive starting points for the discovery of more advanced ALK2 inhibitors

Discovery of Novel Small-Molecule Inhibitors of BRD4 Using Structure-Based Virtual Screening

Bromodomains (BRDs) are epigenetic readers that recognize acetylated-lysine (KAc) on proteins and are implicated in a number of diseases. We describe a virtual screening approach to identify BRD inhibitors. Key elements of this approach are the extensive design and use of substructure queries to compile a set of commercially available compounds featuring novel putative KAc mimetics and docking this set for final compound selection. We describe the validation of this approach by applying it to the first BRD of BRD4. The selection and testing of 143 compounds lead to the discovery of six novel hits, including four unprecedented KAc mimetics. We solved the crystal structure of four hits, determined their binding mode, and improved their potency through synthesis and the purchase of derivatives. This work provides a validated virtual screening approach that is applicable to other BRDs and describes novel KAc mimetics that can be further explored to design more potent inhibitors