Mariko Hashimoto

Discovery of diphenylmethane analogs as anti-bovine diarrhea viral agents.

Based on antiviral screening of our diphenylmethane derivatives prepared as steroid substitutes, we identified a 1,1-diphenylcyclobutane analog (9) and two diethyldiphenylsilane analogs (12 and 13) as superior lead compounds with potent anti-bovine viral diarrhea virus (BVDV) activity, having 50% effective concentration (EC(50): based on reduction of BVDV replication-induced cell destruction) and 50% cytotoxic concentration (CC(50): based on reduction of viable cell number) values of 6.2-8.4 microM and >100 microM, respectively, in Madin-Darby bovine kidney (MDBK) cells infected with BVDV.

Trimeric purine nucleoside phosphorylase: Exploring postulated one-third-of-the-sites binding in the transition state

Transition-state analogue inhibitors, immucillins, were reported to bind to trimeric purine nucleoside phosphorylase (PNP) with the stoichiometry of one molecule per enzyme trimer [Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L. Biochem. 1998, 37, 8615]. In attempts to observe and better understand the nature of this phenomenon we have conducted calorimetric titrations of the recombinant calf PNP complexed with immucillin H. However, by striking contrast to the earlier reports, we have not observed negative cooperativity and we got the stoichiometry of three immucillin molecules per enzyme trimer. Similar results were obtained from fluorimetric titrations, and for other inhibitors bearing features of the transition state. However, we observed apparent cooperativity between enzyme subunits and apparent lower stoichiometry when we used the recombinant enzyme not fully purified from hypoxanthine, which is moped from Escherichia coli cells. Results presented here prove that one-third-of-the-sites binding does not occur for trimeric PNP, and give the highly probable explanation why previous experiments were interpreted in terms of this phenomenon.

Development of tryptase inhibitors derived from thalidomide.

A novel series of tryptase inhibitors with a N-phenylphthalimide skeleton structurally derived from thalidomide (1) has been developed. Structure-activity relationship studies led to a potent and selective tryptase inhibitor, 2-(4-cyanophenyl)isoindole-1,3-dione-5-yl 3-(2-aminopyridin-5-yl)propanoate (7), with the IC50 value of 78 nM.

9-Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow-binding picomolar inhibitors of trimeric purine nucleoside phosphorylase

Genetic deficiency of purine nucleoside phosphorylase (PNP; EC 2.4.2.1) activity leads to a severe selective disorder of T‐cell function. Therefore, potent inhibitors of mammalian PNP are expected to act as selective immunosuppressive agents against, for example, T‐cell cancers and some autoimmune diseases. 9‐(5′,5′‐difluoro‐5′‐phosphonopentyl)‐9‐deazaguanine (DFPP‐DG) was found to be a slow‐ and tight‐binding inhibitor of mammalian PNP. The inhibition constant at equilibrium (1 mm phosphate concentration) with calf spleen PNP was shown to be  = 85 ± 13 pm (pH 7.0, 25 °C), whereas the apparent inhibition constant determined by classical methods was two orders of magnitude higher ( = 4.4 ± 0.6 nm). The rate constant for formation of the enzyme/inhibitor reversible complex is (8.4 ± 0.5) × 105 m−1·s−1, which is a value that is too low to be diffusion‐controlled. The picomolar binding of DFPP‐DG was confirmed by fluorimetric titration, which led to a dissociation constant of 254 pm (68% confidence interval is 147–389 pm). Stopped‐flow experiments, together with the above data, are most consistent with a two‐step binding mechanism: E + I ↔ (EI) ↔ (EI)*. The rate constants for reversible enzyme/inhibitor complex formation (EI), and for the conformational change (EI) ↔ (EI)*, are kon1 = (17.46 ± 0.05) × 105 m−1·s−1, koff1 = (0.021 ± 0.003) s−1, kon2 = (1.22 ± 0.08) s−1 and koff2 = (0.024 ± 0.005) s−1, respectively. This leads to inhibition constants for the first (EI) and second (EI)* complexes of Ki = 12.1 nM (68% confidence interval is 8.7–15.5 nm) and  = 237 pm (68% confidence interval is 123–401 pm), respectively. At a concentration of 10−4 m, DFPP‐DG exhibits weak, but statistically significant, inhibition of the growth of cell lines sensible to inhibition of PNP activity, such as human adult T‐cell leukaemia and lymphoma (Jurkat, HuT78 and CCRF‐CEM). Similar inhibitory activities of the tested compound were noted on the growth of lymphocytes collected from patients with Hashimoto’s thyroiditis and Graves’ disease. The observed weak cytotoxicity may be a result of poor membrane permeability.

Application of a 3,3-diphenylpentane skeleton as a multi-template for creation of HMG-CoA reductase inhibitors

Based on our hypothesis that the 3,3-diphenylpentane (DPP) skeleton is useful as a multi-template for creation of various biologically active compounds and acts as a steroid skeleton substitute, we designed and synthesized novel HMG-CoA reductase inhibitors with a DPP skeleton. Among them, sodium (E,3R,5S)-7-(2-(4-fluorophenyl)-4-(3-phenylpentan-3-yl)phenyl)-3,5-dihydroxy-hept-6-enoate showed potent HMG-CoA reductase-inhibitory activity comparable with that of clinically useful mevastatin.

Overexpressed proteins may act as mops removing their ligands from the host cells: A case study of calf PNP

Calf purine nucleoside phosphorylase (PNP) was overexpressed in Escherichia coli. The basic kinetic parameters of recombinant PNP were found to be similar to the values published previously for non-recombinant PNP from calf spleen. However, upon titration of the recombinant enzyme with the tight-binding multisubstrate analogue inhibitor DFPP-DG, endothermic as well as exothermic signals were obtained. This was not the case for PNP isolated from calf spleen for which only the endothermic process was observed. Further calorimetric titrations of the recombinant and non-recombinant enzyme with its potent and moderate ligands, and studied involving partial inactivation of the enzyme, lead to the conclusion that a part of the recombinant enzyme forms a complex with its product, hypoxanthine, although hypoxanthine was not present at any purification stage except for its natural occurrence in E. coli cells. Binding of hypoxanthine is accompanied with a large negative change of the free enthalpy, and therefore the replacement of this compound by DFPP-DG yields positive heat signal. Our data obtained with calf PNP indicate that similar processes--moping of ligands from the host cells--may take place in the case of other proteins with high overexpression yield.

Thermodynamic studies of interactions of calf spleen PNP with acyclic phosphonate inhibitors

The Gibbs binding energy and entropy/enthalpy contributions to the interaction of calf spleen purine nucleoside phosphorylase (PNP) with the novel multisubstrate analogue DFPP-DG, as well as with DFPP-G and (S)-PMP-DAP were determined by fluorescence and calorimetric studies. Results were compared with findings for guanine - a natural reaction product and inhibitor.

Antiproliferative Activity of Purine Nucleoside Phosphorylase Multisubstrate Analogue Inhibitors Containing Difluoromethylene Phosphonic Acid against Leukaemia and Lymphoma Cells

Potent inhibitors of purine nucleoside phosphorylase (PNP) are expected to act as selective agents against T‐cell tumours. Five compounds with guanine, three with hypoxanthine, and five with 9‐deazaguanine, all connected by a linker with difluoromethylene phosphonic acid, were studied on their inhibitory potential against human and calf PNPs. Antiproliferative activity of these analogues against lymphocytes as well as lymphoma and leukaemia cells has been also investigated. All tested compounds act as multisubstrate analogue inhibitors of PNP with the apparent inhibition constants in the range 5–100 nm, and also show a slight antiproliferative activity. Analogues with 9‐deazaguanine aglycone have better anti‐leukaemic and anti‐lymphoma activities compared to the guanine and hypoxanthine analogues, and applied in the concentration of 100 μm, caused a statistically significant decrease in the cell viability in all human leukaemia and lymphoma cells used. Despite the high PNP inhibitory potential of tested analogues, no differences were observed between the effects on the growth of tumour cells sensible to the inhibition of PNP, such as human adult T‐cell leukaemia and lymphoma cells, and other investigated cells. Obtained poor effects on cell proliferation could be explained probably by a poor ability of tested compounds to penetrate cell membranes.