Identification of AHCY inhibitors using novel high-throughput mass spectrometry

S-adenosylhomocysteine hydrolase (AHCY) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and l-homocysteine. This enzyme is frequently overexpressed in many tumor types and is considered to be a validated anti-tumor target. In order to enable the development of small molecule AHCY inhibitors as targeted cancer therapeutics we developed an assay based on a RapidFire high-throughput mass spectrometry detection system, which allows the direct measurement of AHCY enzymatic activity. This technique avoids many of the problems associate with the previously reported method of using a thiol-reactive fluorescence probes to measure AHCY activity. Screening of a ∼500,000 compound library using this technique identified multiple SAH competitive hits. Co-crystal structures of the hit compounds complexed with AHCY were obtained showing that the compounds indeed bind in the SAH site of the enzyme. In addition, some hit compounds increased the SAH levels in HCT116 cells and showed growth inhibition. These compounds could be promising starting points for the optimization of cancer treatments. •S-adenosylhomocysteine hydrolase (AHCY) is a potential target in many cancers.•A high throughput method was developed to identify novel AHCY inhibitor compounds.•Hit compounds showed S-adenosylhomocysteine (SAH) competitiveness.•They were found to increase SAH levels in HCT116 cells and inhibit their growth.•These compounds could be the basis for new cancer treatments.