Identification of novel small-molecule inhibitors for human transketolase by high-throughput screening with fluorescent intensity (FLINT) assay.

The metabolic enzyme transketolase (TK) plays a crucial role in tumor cell nucleic acid synthesis, using glucose through the elevated nonoxidative pentose phosphate pathway (PPP). Identification of inhibitors specifically targeting TK and preventing the nonoxidative PPP from generating the RNA ribose precursor, ribose-5-phosphate, provides a novel approach for developing effective anticancer therapeutic agents. The full-length human transketolase gene was cloned and expressed in Escherichia coli and the recombinant human transketolase protein purified to homogeneity. A fluorescent intensity (FLINT) assay was developed and optimized. Library compounds were screened in a high-throughput screening (HTS) campaign using the FLINT assay. Fifty-four initial hits were identified. Among them, 2 scaffolds with high selectivity, ideal physiochemical properties, and low molecular weight were selected for lead optimization studies. These compounds specifically inhibited in vitro TK enzyme activity and suppressed tumor cell proliferation in at least 3 cancer cell lines: SW620, LS174T, and MIA PaCa-2. Identification of these active scaffolds represents a good starting point for development of drugs specifically targeting TK and the nonoxidative PPP for cancer therapy.