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

Abstract

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 IC measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC 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.

Adipose triglyceride lipase is regulated by CAMKK2-AMPK signaling and drives advanced prostate cancer

“…Like in cell culture, ATGL KO increased intratumoral levels of glucose and lactate, indicating that blockade of ATGL caused a similar metabolic shift towards glycolysis in vivo ( Figures 7C-D ). Pharmacological inhibition of murine prostate cancer cells with atglistatin also increased ECAR, which could be reversed upon treatment with the glycolysis inhibitor AZ-PFKFB3-26 60,61 ( Figures 7E-F ). Together, these results indicated that targeting ATGL in CRPC creates a metabolic shift towards glycolysis that we hypothesized could be therapeutically exploited.…”

Grouping concentration response curves by features of their shape to aid rapid and consistent analysis of large data sets in high throughput screens

“…A key way forward here is typically a suite of orthogonal biochemical and biophysical assays that run concurrently to identify the true target actives to act as chemical starting points. [13][14] This then leads into warning flags for the suspicious CR output datasets. These can contain a large proportion of perfect curves -those with very tight confidence intervals and slopes of 1 ( Figure 4 G) as well as data sets where most curves have very steep slopes.…”

High-Throughput Mechanism of Inhibition

“…Often, these sorts of downstream triage assays, such as identification of metal contamination or redox activity, that may reveal hits having undesired mechanisms, including so-called PAINs, 6 are increasingly brought in earlier after HTS. 7 Alongside this chemical and physical characterization, it is also desirable to gain an earlier understanding of the mode of interaction between the inhibitor and the target enzyme. 8…”