Differential inhibitor sensitivity between human recombinant and native photoreceptor cGMP-phosphodiesterases (PDE6s).

Human photoreceptor cGMP-phosphodiesterases (PDE6s) are important reagents in PDE inhibitor discovery. However, recombinant human PDE6s have not been expressed, and isolation of native human PDE6s is highly difficult. In this study, the catalytic subunit(s) of human rod and cone PDE6s (PDE6alphabeta and PDE6alpha', respectively) were co-expressed or expressed separately as catalytically active enzymes. Sildenafil inhibited both the recombinant PDE6s in a dose-dependent manner with Ki values of 94 and 98 nM, respectively. These Ki values were four-fold higher than that (25 nM) of a human native PDE6 preparation. Similarly, 3-isobutyl-1-methylxanthine (IBMX)'s Ki values for the recombinant PDE6s were five- to eight-fold higher than that of the native enzyme. However, E4021 and zaprinast exhibited much (30-80-fold) lower potencies for the recombinant PDE6s than for the native enzyme. Additional PDE5 inhibitors representing other structural classes and possessing different selectivity against native PDE6 also showed different potencies against the recombinant and native PDE6s. In particular, one class of xanthine analogues exhibited significantly (5-15-fold) higher potencies for the recombinant PDE6s than for the native enzyme. Our data demonstrates that the recombinant and native PDE6s exhibit differential sensitivity to inhibitors, and cautions the use of recombinant catalytic subunits of PDE6 in drug discovery or in structural/functional studies.

Induction of lysosomal and plasma membrane-bound sialidases in human T-cells via T-cell receptor.

Among the three isoenzymes of neuraminidase (Neu) or sialidase, Neu-1 has been suggested to be induced by cell activation and to be involved in IL (interleukin)-4 biosynthesis in murine T-cells. In the present study, we found that antigen-induced airway eosinophilia, a typical response dependent on Th2 (T-helper cell type 2) cytokines, as well as mRNA expression of Th2 cytokines, including IL-4, are suppressed in Neu-1-deficient mice, thereby demonstrating the in vivo role of murine Neu-1 in regulation of Th2 cytokines. To elucidate the roles of various sialidases in human T-cell activation, we investigated their tissue distribution, gene induction and function. Neu-1 is the predominant isoenzyme at the mRNA level in most tissues and cells in both mice and humans, including T-cells. T-cells also have significant levels of Neu-3 mRNAs, albeit much lower than those of Neu-1, whereas the levels of Neu-2 mRNAs are minimal. In human T-cells, both Neu-1 and Neu-3 mRNAs are significantly induced by T-cell-receptor stimulation, as is sialidase activity against 4-methylumbelliferyl- N -acetylneuramic acid (a substrate for both Neu-1 and Neu-3) and the ganglioside G(D1a) [NeuAcalpha2-3Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-cer] (a substrate for Neu-3, but not for Neu-1). The expression of the two sialidase genes may be under differential regulation. Western blot analysis and enzymic comparison with recombinant sialidases have revealed that Neu-3 is induced as a major isoform in activated cells. The induction of Neu-1 and Neu-3 in T-cells is unique. In human monocytes and neutrophils stimulated with various agents, the only observation of sialidase induction has been by IL-1 in neutrophils. Functionally, a major difference has been observed in Jurkat T-cell lines over-expressing Neu-1- and Neu-3. Upon T-cell receptor stimulation, IL-2, interferon-gamma, IL-4 and IL-13 are induced in the Neu-1 line, whereas in the Neu-3 line the same cytokines are induced, with the exception of IL-4. Taken together, these results suggest an important immunoregulatory role for both Neu-1 and Neu-3 in humans.

Identification and characterization of a new human type 9 cGMP-specific phosphodiesterase splice variant (PDE9A5). Differential tissue distribution and subcellular localization of PDE9A variants.

Previously, four splice variants of human cGMP-specific phosphodiesterase (PDE) 9A (PDEs 9A1, 9A2, 9A3 and 9A4) have been identified. In this study, we have cloned a cDNA representing a new human PDE9A variant (PDE9A5). PDE9A5 encodes a protein of 492 amino acids, smaller than PDEs 9A1 and 9A2 but larger than PDEs 9A3 and 9A4. The exon structure of PDE9A5 is different from those of PDEs 9A1, 9A2, 9A3 and 9A4 in that, of the 20 exons of PDE9A gene, it lacks exons 2 and 5. PDE9A5 has been characterized in comparison with PDE9A1, the longest PDE9A variant. PDEs 9A5 and 9A1 have similar enzymatic properties. They both have a high affinity for cGMP with similar Km values (0.39 and 0.25 microM, respectively), although they have slightly different Vmax values (2.55 and 0.96 micromol/min/mg, respectively). They exhibit very similar divalent metal ion dependency and inhibitor sensitivity. Real-time quantitative PCR analysis shows that PDEs 9A5 and 9A1 exhibit differential tissue distribution. They are highly expressed in immune tissues (spleen, lymph node and thymus) and are more abundant in T cells than in B cells, neutrophils and monocytes. When transiently expressed in HEK293 cells, PDEs 9A5 and 9A1 proteins exhibit differential subcellular localization. PDE9A5 localizes exclusively in the cytoplasm, whereas PDE9A1 localizes in the nucleus only. The nuclear localization of PDE9A1 is dependent on a unique pat7 motif. By Western blot analysis, native PDE9A1 is detectable in the nucleus but not in the cytoplasm of T cells. Thus, to our knowledge, PDE9A1 is the only PDE isoform found to localize exclusively in the nucleus. We speculate that the physiological role of the PDE9A diversity may be imparting cGMP-metabolizing ability to specific cellular compartments in appropriate tissues.

Identification of full, partial and inverse CC chemokine receptor 3 agonists using [35S]GTPgammaS binding.

Study of the CC chemokine receptor 3 (CCR3) has been limited to using radiolabeled agonist chemokines. A small molecule CCR3 antagonist, 2-[(6-amino-2-benzothiazolyl)thio]-N-[1-[(3,4-dichlorylphenyl)methyl]-4-piperidinyl]acetamide, Banyu (I), was tritiated and used for pharmacological studies. Banyu (I) has a K(d) of 5.0+/-0.4 and 4.3+/-1.8 nM on human CCR3 transfectants and eosinophils, and noncompetitively inhibits [125I]eotaxin binding and eotaxin-induced [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. The proportion of [125I]eotaxin: [3H]Banyu (I) binding sites in eosinophils or transfectants was 35% or 13%, although both binding sites were overexpressed in transfectants. CCR3 spontaneously couples to G-proteins in CCR3 transfectants, demonstrated by changes in basal and eotaxin-induced [35S]GTPgammaS binding under reduced NaCl and GDP concentrations. Consequently, Banyu (I) was identified as an inverse agonist. In contrast, CCL18 and I-TAC (interferon-inducible T cell alpha-chemoattractant) were neutral antagonists, inhibiting eotaxin-induced [35S]GTPgammaS binding, with minimal effect on basal coupling of CCR3 to G proteins. Eotaxin, eotaxin-2 and monocyte chemoattractant protein (MCP)-4 are full agonists inducing [35S]GTPgammaS binding; eotaxin-3, MCP-3, RANTES (regulated on activation normal T cell expressed and secreted), vMIP-I (Kaposi's sarcoma-associated herpesvirus macrophage inflammatory protein-) and vMIP-II are partial agonists, indicating that this is a sensitive method to quantitate agonist efficacy.

Pharmacology of N-(3,5-dichloro-1-oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinoline carboxamide (SCH 351591), a novel, orally active phosphodiesterase 4 inhibitor.

N-(3,5-Dichloro-1-oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinoline carboxamide (SCH 351591) has been identified as a potent (IC(50) = 58 nM) and highly selective type 4 phosphodiesterase (PDE4) inhibitor with oral bioactivity in several animal models of lung inflammation. N-(3,5-Dichloro-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinoline carboxamide (SCH 365351), the only significant in vivo metabolite, is also a potent and highly selective PDE4 inhibitor (IC(50) = 20 nM). Both SCH 351591 and SCH 365351 inhibited cytokine production in human blood mononuclear cell preparations. Oral SCH 351591 significantly attenuated allergen-induced eosinophilia and airway hyperreactivity in allergic guinea pigs at doses as low as 1 mg/kg. In this model, oral SCH 365351 showed similar potency. When SCH 351591 was administered orally to allergic cynomolgus monkeys at 3 mg/kg, Ascaris suum-induced lung eosinophilia was blocked. Hyperventilation-induced bronchospasm in nonallergic guinea pigs, a model for exercise-induced asthma, was also suppressed significantly by oral SCH 351591 at 0.3 mg/kg. Cilomilast (SB 207499; Ariflo), a PDE4 inhibitor currently being developed for asthma and chronic obstructive pulmonary disease (COPD), was 10- to 30-fold less potent than SCH 351591 at inhibiting guinea pig lung eosinophilia and hyperventilation-induced bronchospasm. In a ferret model of emesis, maximum nonemetic oral doses of SCH 351591 and cilomilast were 5 and 1 mg/kg, respectively. Comparison of plasma levels at these nonemetic doses in ferrets to those at doses inhibiting hyperventilation-induced bronchospasm in guinea pigs gave a therapeutic ratio of 16 for SCH 351591 and 4 for cilomilast. Thus, SCH 351591 exhibits a promising preclinical profile as a treatment for asthma and COPD.