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We have investigated various nitric oxide (NO) synthase inhibitors for their affinity and selectivity toward the three human isoenzymes in radioligand binding experiments. Therefore, we developed the new radioligand [(3)H]2-amino-4-picoline to measure binding of these compounds to the three human NO synthase (NOS) isoenzymes. Aminopicoline is a potent and nonselective inhibitor of all three isoforms. [(3)H]2-amino-4-picoline bound saturably and with high affinity to human NOSs. Affinity constants (K(D) values) of 59, 111, and 136 nM were obtained for the inducible, neuronal, and endothelial NOS isoforms (iNOS, nNOS, eNOS). Binding of [(3)H]2-amino-4-picoline was competitive with the substrate arginine. From all the inhibitors tested, AMT (2-amino-5, 6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride) showed the highest affinity and no selectivity. L-NIL [L-N(6)-(1-Iminoethyl)lysine hydrochloride] and aminoguanidine were moderately iNOS-selective while L-NA (N(G)-nitro-L-arginine) and L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride) showed selectivity toward the constitutive isoforms. High iNOS versus eNOS selectivity was found for 1400W, whereas several isothiourea derivatives and 1400W displayed moderate n- versus eNOS selectivity. To relate the affinity of these compounds to their inhibitory potency, we measured the inhibitory potency under almost identical conditions using a new microtiter plate assay. The inhibitory potency of selective and nonselective NOS inhibitors was almost exactly mirrored by their affinity toward the different isoenzymes. Highly significant correlations were obtained between the potency of enzyme inhibition and the inhibition of [(3)H]2-amino-4-picoline binding for all three isoenzymes. These data show that the potency and selectivity of NOS inhibitors are solely determined by their affinity toward the different isoforms. Furthermore, these data identify the new radioligand [(3)H]2-amino-4-picoline as a very useful radiolabel for the investigation of the substrate binding site of all three isoforms.

Nitric oxide (NO) is a key physiological mediator and disturbed regulation of NO release is associated with the pathophysiology of almost all inflammatory diseases. A multitude of inhibitors of NOSs (nitric oxide synthases) have been developed, initially with low or even no selectivity against the constitutively expressed NOS isoforms, eNOS (endothelial NOS) and nNOS (neuronal NOS). In the meanwhile these efforts yielded potent and highly selective iNOS (inducible NOS) inhibitors. Moreover, iNOS inhibitors have been shown to exert beneficial anti-inflammatory effects in a wide variety of acute and chronic animal models of inflammation. In the present mini-review, we summarize some of our current knowledge of inhibitors of the iNOS isoenzyme, their biochemical properties and efficacy in animal models of pulmonary diseases and in human disease itself. Moreover, the potential benefit of iNOS inhibition in animal models of COPD (chronic obstructive pulmonary disease), such as cigarette smoke-induced pulmonary inflammation, has not been explicitly studied so far. In this context, we demonstrated recently that both a semi-selective iNOS inhibitor {l-NIL [N6-(1-iminoethyl)-l-lysine hydrochloride]} and highly selective iNOS inhibitors (GW274150 and BYK402750) potently diminished inflammation in a cigarette smoke mouse model mimicking certain aspects of human COPD. Therefore, despite the disappointing results from recent asthma trials, iNOS inhibition could still be of therapeutic utility in COPD, a concept which needs to be challenged and validated in human disease.

The solution structure of a recombinant mutant [rSP‐C (FFI)] of the human surfactant‐associated protein C (hSP‐C) in a mixture of chloroform and methanol was determined by high‐resolution NMR spectroscopy. rSP‐C (FFI) contains a helix from Phe5 to the C‐terminal Leu34 and is thus longer by two residues than the helix of porcine SP‐C (pSP‐C), which is reported to start at Val7 in the same solvent. Two sets of resonances at the C‐terminus of the peptide were observed, which are explained by low‐order oligomerization, probably dimerization of rSP‐C (FFI) in its α‐helical form. The dimerization may be induced by hydrogen bonding of the C‐terminal carboxylic groups or by the strictly conserved C‐terminal heptapeptide segment with a motif similar to the GxxxG dimerization motif of glycophorin A. Dimerization at the heptapeptide segment would be consistent with findings based on electrospray ionization MS data, chemical cross‐linking studies, and CNBr cleavage data.

Human P-glycoprotein (P-gp), an integral membrane transport protein, is responsible for the efflux of various drugs, including cytostatics from cancer cells leading to multidrug resistance. P-gp is composed of two homologous half domains, each carrying one nucleotide binding site. The drug extrusion is ATP-dependent and can be inhibited by chemosensitizers, such as the dihydropyridine derivative dexniguldipine-HCl, through direct interaction with P-gp. To evaluate the mechanism(s) of chemosensitization and identify the binding sites of dexniguldipine-HCl, a tritium-labeled azido analog of dexniguldipine, [3 H]B9209-005, was used as a photoaffinity probe. Using the multidrug resistant T-lymphoblastoid cell line CCRF-ADR5000, two proteins were specifically labeled in membranes by [3 H]B9209-005. These proteins were identified by immunoprecipitation such as P-gp and its N-terminal fragment. The membranes were solubilized and the labeled P-gp proteins first isolated by lectin-chromatography and then digested with trypsin. SDS-polyacrylamide gel electrophoresisanalysis of the digest revealed a major radioactive 7-kDa fragment. The tryptic fragments were separated by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS). The MS results, corroborated by MALDI-MS of peptides after one step of Edman analysis, identified the radioactive 7-kDa band as the dexniguldipine-bound, tryptic P-gp peptide, 468 -527. This sequence region is flanked by the Walker motifs A and B of the N-terminal ATP-binding cassette suggesting direct interaction of the chemosensitizer with the nucleotide binding site is involved in the mechanism of chemosensitization.Tumor cells in vitro and in vivo can develop simultaneous resistance to the lethal effects of a variety of cytotoxic drugs (Endicott and Ling, 1989). This so-called multidrug resistance (MDR) is a major limiting factor for the efficacy of cancer chemotherapy. Currently, a variety of mechanisms are known that can lead to drug resistance, including reduced cellular drug accumulation, increased detoxification, intracellular vesicularization of drugs, altered enzymatic activities, up-or down-regulation of targets, and enhanced DNA repair (Hayes and Wolf, 1990). One important resistance mechanism is the transport out of cancer cells of chemically-unrelated cytotoxic drugs (such as anthracylines, Vinca alkaloids, colchicine, and taxanes) by the integral membrane phosphoglycoprotein P-glycoprotein (P-gp) under ATP hydrolysis, resulting in low and ineffective intracellular drug concentrations (Gottesman and Pastan, 1993). Although considerable progress has been made during the last few years, the mechanism of recognition and transport of such a broad spectrum of compounds is still poorly understood.Reversal of multidrug resistance is of major clinical interest, and MDR-reversing agents called chemosensitizers have been intensively investigated (Raderer and Scheithauer, 1993;Sikic, 1993). The capability of reversi...