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The 3,5-dichlorobenzoyl-substituted 2-deoxy-d-ribose-1-phosphate derivative, designated Cf2891, was found to inhibit a variety of pyrimidine and purine nucleoside phosphorylases (NPs) with preference for uridine- and inosine-hydrolyzing enzymes [uridine phosphorylase (UP; EC 18.104.22.168), pyrimidine nucleoside phosphorylase (PyNP; EC 22.214.171.124) and purine nucleoside phosphorylase (PNP; EC 126.96.36.199)]. Kinetic analyses revealed that Cf2891 competes with inorganic phosphate (Pi) for binding to the NPs and, depending on the nature of the enzyme, acts as a competitive or non-competitive inhibitor with regard to the nucleoside binding site. Also, the compound prevents breakdown of pyrimidine analogues used in the treatment of viral infections and cancer. Since NPs are abundantly present in tumor tissue and may be overexpressed due to secondary bacterial infections in immunocompromised patients suffering viral infections, Cf2891 may serve as a lead molecule for the development of inhibitors to be used in nucleoside-based combination therapy.