A high-throughput screening triage workflow to authenticate a novel series of PFKFB3 inhibitors

DOI: 10.1177/2472555217732289

Authors: Stephen A. St-Gallay (AstraZeneca) , Neil Bennett (AstraZeneca) , Susan E. Critchlow (AstraZeneca) , Nicola Curtis (AstraZeneca) , Gareth Davies (AstraZeneca) , Judit Debreczeni (AstraZeneca) , Nicola Evans (Cancer Research UK Therapeutic Discovery Laboratories) , Ian Hardern (AstraZeneca) , Geoff Holdgate (AstraZeneca) , Neil P. Jones (Cancer Research UK Therapeutic Discovery Laboratories) , Lindsey Leach (AstraZeneca) , Sarita Maman (Cancer Research UK Therapeutic Discovery Laboratories) , Sheila Mcloughlin (Cancer Research UK Therapeutic Discovery Laboratories) , Marian Preston (AstraZeneca) , Laurent Rigoreau (Cancer Research UK Therapeutic Discovery Laboratories) , Andrew Thomas (AstraZeneca) , Andrew P. Turnbull (Cancer Research UK Therapeutic Discovery Laboratories) , Graeme Walker (AstraZeneca) , Jarrod Walsh (AstraZeneca) , Richard A. Ward (AstraZeneca) , Ed Wheatley (Cancer Research UK Therapeutic Discovery Laboratories) , Jon Winter-Holt (AstraZeneca)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Slas Discovery: Advancing Life Sciences R&d , VOL 27

State: Published (Approved)
Published: September 2017
Diamond Proposal Number(s): 5735

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 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.

Journal Keywords: HTS; isothermal calorimetry; sigma hole; Warburg effect; PAINS

Subject Areas: Biology and Bio-materials, Medicine


Instruments: I03-Macromolecular Crystallography

Added On: 06/10/2017 11:40

Discipline Tags:

Non-Communicable Diseases Health & Wellbeing Cancer Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)