A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors.

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.

Molecular basis of USP7 inhibition by selective small-molecule inhibitors.

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

Structure-Based Design of Potent and Selective Inhibitors of the Metabolic Kinase PFKFB3.

A weak screening hit with suboptimal physicochemical properties was optimized against PFKFB3 kinase using critical structure-guided insights. The resulting compounds demonstrated high selectivity over related PFKFB isoforms and modulation of the target in a cellular context. A selected example demonstrated exposure in animals following oral dosing. Examples from this series may serve as useful probes to understand the emerging biology of this metabolic target.