Sadao Hikishima

Acid sphingomyelinase inhibition suppresses lipopolysaccharide-mediated release of inflammatory cytokines from macrophages and protects against disease pathology in dextran sulphate sodium-induced colitis in mice

Lipopolysaccharide (LPS) and inflammatory cytokines cause activation of sphingomyelinases (SMases) and subsequent hydrolysis of sphingomyelin (SM) to produce a lipid messenger ceramide. The design of SMase inhibitors may offer new therapies for the treatment of LPS‐ and cytokine‐related inflammatory bowel disease. We synthesized a series of difluoromethylene analogues of SM (SMAs). We report here the effects of the most potent SMase inhibitor, SMA‐7, on the LPS‐mediated release of tumour necrosis factor‐α, interleukin‐1β and interleukin‐6 from THP‐1 macrophages and the pathology of dextran sulphate sodium (DSS)‐induced colitis in mice. SMA‐7 suppressed the LPS‐induced cytokine release and nuclear factor‐κB activation. LPS stimulation caused a four‐fold increase in acid SMase activation, but little increase in neutral SMase activity. The presence of 10 μm SMA‐7 caused acid SMase to remain at the control levels and reduced the formation of ceramide. HT‐29 cells had significantly decreased cell viability when incubated with media from LPS‐stimulated THP‐1 macrophages. However, incubating the colon cells in media from both SMA‐7 and LPS‐treated macrophages caused little decrease in viability, suggesting that ceramide has a role in the LPS‐stimulated signalling that releases cytotoxic factors against colon cells. Oral administration of SMA‐7 to mice with 2% DSS in the drinking water, for 10 or 21 consecutive days, reduced significantly the cytokine levels in the colon and the severity of colonic injury. These findings suggest a central role for acid SMase/ceramide signalling in the pathology of DSS‐induced colitis in mice, indicating a possible preventive or therapeutic role for SMase inhibitor in inflammatory bowel disease.

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.

Diastereoselective Synthesis of γ-Amino-δ-hydroxy-α,α-difluorophosphonates: A Vehicle for Structure― activity Relationship Studies on SMA-7, a Potent Sphingomyelinase Inhibitor

A highly diastereoselective synthesis of 2-amino alcohol derivatives bearing a difluoromethylphosphonothioate group at the 3-position was achieved through LiAlH(O-t-Bu)(3)-mediated reduction of the corresponding alpha-amino ketones. The phosphonothioate moiety of the product was readily converted into the corresponding phosphonate by oxidation with m-CPBA, followed by aqueous workup. The developed methods should be useful for SAR studies of SMA-7, a potent inhibitor of SMases.

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.

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.

Inhibitory Properties of Nucleotides with Difluoromethylenephosphonic Acid as a Phosphate Mimic versus Calf Spleen Purine Nucleoside Phosphorylase and Effect of These Analogues on the Viability of Human Blood Lymphocytes

Several cyclic and acyclic 6-keto purine nucleotides with difluoromethylenephosphonic acid as phosphate mimic are proved to be potent inhibitors of mammalian purine nucleoside phosphorylase (PNP). Antiproliferative activity of these analogues on the growth of human blood lymphocytes was tested by MTT assay. Compared to inhibitory effects on the growth of human blood T-lymphocytes isolated from healthy donors, all analogues significantly slow down proliferation of T-lymphocytes isolated from patients with autoimmune thyroid disease—Hashimotos thyroiditis.

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.