Di-, tri- and tetra-5'-O-phosphorothioadenosyl substituted polyols as inhibitors of Fhit: Importance of the α-β bridging oxygen and β phosphorus replacement

Here we link adenosine 5'-O-phosphates and phosphorothioates to short-chain polyols to generate a series of substrate analogs. We obtain structure-activity data in the form of in vitro Fhit inhibition for four types of analog substitutions and describe two compounds, inhibitory constants for which are 65 and 75-fold lower than natural substrates.The best Fhit inhibitors obtained to date separate two or more 5'-O-phosphoromonothioadenosyl moieties with as many bond lengths as in AppppA, maintain oxygen at the location of the α-β bridging oxygen, and replace carbon for the β phosphorus.Loss of Fhit protein is among the earliest known events in the development of a variety of the most common and lethal human malignancies [1]. Loss of Fhit leads to cells that are deficient in programmed cell death and that form tumors in mice while Fhit reexpression in Fhit-cancer cells reduces tumorigenicity and restores programmed cell death [2-6]. Infection of Fhit +/- mice with viruses that re-express Fhit reduce cancer occurrence [7], apparently by killing pre-neoplastic cells that have lost the wild-type Fhit allele. Human Fhit protein, a member of the Fhit branch of the histidine triad superfamily of nucleotide-binding proteins, binds and hydrolyzes diadenosine polyphosphates such as ApppA and AppppA (1) into AMP plus ADP and ATP, respectively [8-10]. His96, which is responsible for covalent catalysis and more than 4× 106-fold of rate enhancement in ApppA hydrolysis [9,11-13], is nonetheless dispensable for ApppA-binding and tumor suppression, suggesting that Fhit function in tumor suppression depends on formation of an E-S complex [2,12]. If Fhit-substrate complexes promote tumor suppression by stimulating a pro-apoptotic effector, then Fhit inhibitors that resemble natural substrates may promote Fhit function. Similarly, Fhit inhibitors with normative features may antagonize Fhit function. Either class of compounds may be important in dissecting Fhit cell biology and regulating