Efficacy of Climate Forcings in PDRMIP Models.

Quantifying the efficacy of different climate forcings is important for understanding the real-world climate sensitivity. This study presents a systematic multimodel analysis of different climate driver efficacies using simulations from the Precipitation Driver and Response Model Intercomparison Project (PDRMIP). Efficacies calculated from instantaneous radiative forcing deviate considerably from unity across forcing agents and models. Effective radiative forcing (ERF) is a better predictor of global mean near-surface air temperature (GSAT) change. Efficacies are closest to one when ERF is computed using fixed sea surface temperature experiments and adjusted for land surface temperature changes using radiative kernels. Multimodel mean efficacies based on ERF are close to one for global perturbations of methane, sulfate, black carbon, and insolation, but there is notable intermodel spread. We do not find robust evidence that the geographic location of sulfate aerosol affects its efficacy. GSAT is found to respond more slowly to aerosol forcing than CO2 in the early stages of simulations. Despite these differences, we find that there is no evidence for an efficacy effect on historical GSAT trend estimates based on simulations with an impulse response model, nor on the resulting estimates of climate sensitivity derived from the historical period. However, the considerable intermodel spread in the computed efficacies means that we cannot rule out an efficacy-induced bias of ±0.4 K in equilibrium climate sensitivity to CO2 doubling when estimated using the historical GSAT trend.

Possible impacts of a future grand solar minimum on climate: Stratospheric and global circulation changes.

A future decline in solar activity would not offset projected global warmingA future decline in solar activity could have larger regional effects in winterTop-down mechanism contributes to Northern Hemisphere regional response.

Arylacetamides as peripherally restricted kappa opioid receptor agonists.

Analogues of the kappa (kappa) opioid receptor agonist, ICI 199441, were prepared. Ki values for these analogues at the cloned human kappa opioid receptor ranged from 0.058 to 25 nM. Trifluoromethylaryl derivatives were potent analgesics when administered subcutaneously in the rat and were more peripherally restricted than the parent compound, ICI 199441.

[Pro(3)]Dyn A(1-11)-NH(2): a dynorphin analogue with high selectivity for the kappa opioid receptor.

A proline scan at positions 2 and 3 of the opioid peptide dynorphin A(1-11)-NH(2) led to the discovery of the analogue [Pro(3)]Dyn A(1-11)-NH(2). This analogue possesses high affinity and selectivity for the kappa opioid receptor (K(i)(kappa) = 2.7 nM, K(i) ratio kappa/micro/delta = 1/2110/3260). The gain in selectivity is achieved through an overall reduction of opioid receptor affinity which is most pronounced at micro and delta receptors. The Pro(3) analogue exhibits antagonist properties. Despite its high kappa affinity, [Pro(3)]Dyn A(1-11)-NH(2) is a relatively weak antagonist in both the [(35)S]GTPgammaS assay (IC(50) = 380 nM) and the guinea pig ileum assay (K(e) = 244 nM). Discrepancies between GPI and binding assay have often been ascribed to differential kappa receptor subtypes prevailing in central vs peripheral neurons. Since the [(35)S]GTPgammaS assay uses the same membrane preparations as the binding assay, differential kappa subtypes can be ruled out as an explanation in this case, and the observed behavior rather seems to reflect an intrinsic property of the ligand.

Determination of loperamide in rat plasma and bovine serum albumin by LC.

A rapid, isocratic liquid chromatographic (LC) method was developed for the determination of loperamide (Lop) in solutions of bovine serum albumin (BSA) and rat plasma. Prior to LC analysis, BSA solutions or rat plasma samples were treated with metaphosphoric acid to precipitate protein. Supernatant was directly injected onto a C18 reverse phase column and loperamide was monitored by a UV detector set at 195 nm. The concentrations of Lop in both rat plasma and BSA solution samples were determined by comparison with their calibration curves, which were generated from the peak area ratio of Lop to internal standard, clomipramine versus loperamide concentration. The calibration curves were linear in the range 0-3.0 microg ml(-1) of Lop for the BSA solution sample and 0-1.0 microg ml(-1)for the rat plasma sample. Overall recoveries of loperamide added to BSA and rat plasma samples were 101.4 and 95.5%, respectively. The method is simple (no extraction), rapid (22 min separation time), sensitive (the detection limit of loperamide is 50 ng ml(-1) for the BSA solution sample and 100 ng ml(-1) for the rat plasma sample), reproducible (within-day R.S.D. of 2.59-7.11%, among-day R.S.D. of 1.25-5.97%), and suitable for routine analysis of loperamide in rat plasma and BSA solution samples.

Mechanism of inhibition of human leucocyte elastase by beta-lactams. 2. Stability, reactivation kinetics, and products of beta-lactam-derived E-I complexes.

The monocyclic beta-lactams reported by Knight et al. [Knight, W. B., et al. (1992) Biochemistry 31, 8160; Chabin, R., et al. (1993) Biochemistry 32, 8970] as inhibitors of human leucocyte elastase (HLE) produce stable HLE-inhibitor complexes that slowly reactivate with half-lives ranging from less than 1 to 15 h at 37 degrees C. The complexes produced between PPE and two C-3 dimethyl-substituted beta-lactams are less stable than those produced between HLE and analogous C-3 diethyl-substituted lactams. The stability of the HLE-I complexes is governed primarily by the structure of the substituted urea portion of the inhibitors and not by the identity or presence of a leaving group at C-4 of the lactam ring. In some cases substitutions on the urea portion of the inhibitors yielded complexes that displayed biphasic reactivation kinetics. This suggests the presence of at least two different complexes. The stereochemistry of the leaving group at C-4 has a small effect on the stability of the final complex (1.3-2-fold); therefore, the identity of the final complex is dependent upon the initial stereochemistry at that position. The stability of the complexes was relatively insensitive to hydroxylamine, which suggests that the acyl-enzymes are protected from nucleophilic "rescue". The rate of reactivation of the complex derived from L-680,833,[S-R*,S*)]-4-[(1-(((1-(4- methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-oxo-4-azetidinyl)ben zeneacetic acid, was pH independent, while the L-684,481, (R)-(1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-azeti din one generated complex displayed a pH-dependent reactivation rate. In the latter case, the increase in reactivation rate with pH displayed a pKa of 7.2. This is consistent with the requirement for base catalysis by the active site histidine to regenerate enzymatic activity. Reactivation of the L-680,833-derived complex produced different products as a function of pH, suggesting two different pH-dependent routes of reactivation. At low pH a route that produced primarily the substituted urea is favored, while at higher pH production of two six-membered ring diastereomers competes with urea generation. Thus, the apparent pH independence of the return of activity is the result of two offsetting pathways. Other compounds such as L-670,258, (S)-4-[((((2-naphthylmethyl)amino)carbonyl)-3,3-diethyl-4-oxo-2- azetidinyl)oxy]benzoic acid, reactivate by these two routes as well as by aminolysis by the other urea nitrogen to produce an additional regioisomer. The temperature dependence of the reactivation of the complexes derived from L-684,481 and L-680,833 suggests different mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Damnacanthal is a highly potent, selective inhibitor of p56lck tyrosine kinase activity.

Damnacanthal, an anthraquinone isolated from a plant extract, was found to be a potent, selective inhibitor of p56lck tyrosine kinase activity. The structure, potency, and selectivity of damnacanthal were confirmed by independent synthesis and testing. Damnacanthal exhibited an IC50 of 17 nM for inhibition of p56lck autophosphorylation and an IC50 of 620 nM for phosphorylation of an exogenous peptide by p56lck. Damnacanthal had > 100-fold selectivity for p56lck over the serine/threonine kinases, protein kinase A and protein kinase C, and > 40-fold selectivity for p56lck over four receptor tyrosine kinases. It also demonstrated modest (7-20-fold), but highly statistically significant, selectivity for p56lck over the homologous enzymes p60src and p59fyn. Mechanistic studies demonstrated that damnacanthal was competitive with the peptide binding site, but mixed noncompetitive with the ATP site. Although damnacanthal contains a potentially reactive aldehyde moiety, equilibrium dialysis experiments demonstrated that significant amine formation between damnacanthal and amines occurred only at high concentrations of reactants. However, damnacanthal appeared to bind nonspecifically to membrane lipids and was not active in whole cell tyrosine kinase assays. Damnacanthal is the most potent, selective inhibitor of p56lck tyrosine kinase activity described to date and may represent the starting point for the identification of novel, selective inhibitors of p56lck which are active in whole cell as well as in cell-free systems.

Orally active beta-lactam inhibitors of human leukocyte elastase. 3. Stereospecific synthesis and structure-activity relationships for 3,3-dialkylazetidin-2-ones.

The stereospecific synthesis of several 4-[(4-carboxyphenyl)oxy]- 3,3-dialkyl-1-[[(1-phenylalkyl)-amino]carbonyl]azetidin-2-on es 3 is described in which the C-3 alkyl groups were varied from methyl to butyl as well as allyl, benzyl and methoxymethyl. The structure-activity relations for these compounds are discussed in terms of the hydrolytic stability of the beta-lactam ring, their in vitro inhibitory potency for human leukocyte elastase (HLE), and their in vivo oral efficacy in an HLE-mediated hamster lung hemorrhage assay. Further alkyl substitution on the benzylic urea moiety, especially in the R configuration, afforded enhanced HLE inhibition and in vivo efficacy. The stereochemical assignments for (3R,4S)-4-[(4-carboxyphenyl)oxy]-3-ethyl-3-methyl-1-[[((R)-1- phenylpropyl)amino]carbonyl]azetidin-2-one (42a) (kobs/[I] = 91,000 M-1 s-1) were confirmed with an X-ray structure determination, which was also utilized to develop an HLE inhibition model.

Mechanism of inhibition of human leucocyte elastase by monocyclic beta-lactams.

The kinetic and catalytic mechanisms of time-dependent inhibition of human polymorphonuclear leukocyte elastase (HLE) by the monocyclic beta-lactams described by Knight et al. [Knight, W.B., et al. (1992) Biochemistry 31, 8160] are investigated in this work. The dependence of the pseudo-first-order rate constant (k(obs)) on inhibitor concentration was saturable. The individual kinetic constants for the inhibition by L-680,833, [S-(R*,S*)]-4-[(1-(((1-(4- methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-4-oxo-2- azetidinyl)oxy]benzeneacetic acid, and L-683,845, [S-(R*,S*)]-4-[(1-(((1-(5-benzofuranyl)butyl)amino)carbonyl)- 3,3-diethyl-4-oxo-2-azetidinyl)oxy]benzeneacetic acid, at pH 7.5 were k(inact) = 0.08 and 0.06 s-1 and Ki = 0.14 and 0.06 microM, respectively. The relative potency of this class of compounds as measured by k(inact)/Ki is primarily controlled by the Ki, term which ranged from 6 nM to 8 mM, while K(inact) was relatively insensitive to structural changes and varied by only an order of magnitude. Inactivation by the beta-lactams was efficient, requiring only 1.3 and 1.7 equiv of L-680,833 and L-683,845 to inactivate HLE. These values are indicative of some partitioning between turnover of inhibitor and inactivation. The partition ratio ranged as high as 3.5:1 depending upon the structure of the inhibitors, but this ratio was essentially independent of the availability and identity of a leaving group at C-4 of the lactam ring. Inactivation and partitioning liberate the leaving group when present at C-4. p-Hydroxy-m-nitrophenylacetic acid is liberated from this position at a rate similar to that for enzyme inactivation, suggesting kinetic competence of this process. Other products observed during the interaction of L-680,833 with HLE include a substituted urea, a species previously observed during the base-catalyzed decomposition of this class of compounds, and small amounts of products observed during reactivation of beta-lactam-derived HLE-I complexes. Both the pH dependence of k(inact)/Ki for the inactivation of HLE by [S-(R*,S*)]-4-[(1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl - 4-oxo-2-azetidinyl)oxyl]benzoic acid and V/K for HLE-catalyzed substrate hydrolysis indicate that a single ionizable group with a pK of approximately 7 must be deprotonated for both processes. This group is likely the active site histidine. The data are consistent with initial formation of a Michaelis complex, acylation of the catalytic serine, and loss of the leaving group at C-4 of the original beta-lactam ring followed by partitioning between regeneration of active enzyme and production of a stable enzyme-inhibitor complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Orally active beta-lactam inhibitors of human leukocyte elastase. 2. Effect of C-4 substitution.

The effect of changing the C-4 substituent of 3,3-diethyl-1-[(benzylamino)carbonyl]-2-azetidinone on inhibition of HLE and in a model of HLE-induced lung damage in hamsters was explored. Substituents at this position do not appear to interact strongly with HLE with the most potent compounds having k(obs)/[I] = 6900 M-1 s-1. However, substituents at this position had a marked effect on in vivo activity. The greatest oral activity in the lung hemorrhage assay was achieved with C-4 aryl carboxylic acid ethers (60-85% inhibition at 30 mg/kg po). Based upon the established mechanism of inhibition by these compounds, the C-4 substituent would be released, and therefore, the pharmacological potential of these C-4 substituents was of considerable concern. Fortunately, compounds containing 4-hydroxybenzoic acid and 4-hydroxyphenylacetic acid ethers at C-4 were among the most active analogs. These phenolic acids are also found as urinary metabolites in healthy humans. Other heteroaryls at C-4 were also orally active in this model despite relatively modest enzyme activity.

Specificity, stability, and potency of monocyclic beta-lactam inhibitors of human leucocyte elastase.

Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket.

Mechanism of inhibition of human leukocyte elastase by two cephalosporin derivatives.

The cephalosporin derivatives L 658758 [1-[[3-(acetoxymethyl)-7 alpha-methoxy-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-en-2-yl]carbonyl]proline S,S-dioxide] and L 659286 [1-[[7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo- 1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-aza-(6R)-bicyclo[4.2.0]-o ct-2-en-2-yl]carbonyl]pyrrolidine S,S-dioxide] are mechanism based inhibitors of human leukocyte elastase (HLE). The mechanism involves initial formation of a Michaelis complex followed by acylation of the active site serine. The group on the 3'-methylene is liberated during the course of these reactions, followed by partitioning of an intermediate between hydrolysis to regenerate active enzyme and further modification to produce a stable HLE-inhibitor complex. The partition ratio of 2.0 obtained for the reaction with L 658758 approaches that of an optimal inhibitor. These compounds are functionally irreversible inhibitors as the recovery of activity after inactivation is slow. The half-lives at 37 degrees C of the L 658758 and L 659286 derived HLE-I complexes were 9 and 6.5 h, respectively. The complexes produced by both inhibitors are similar chemically since the thermodynamic parameters for activation to regenerate active enzyme are essentially identical. The free energy of activation for this process is dominated primarily by the enthalpy term. The stability of the final complexes likely arises from Michael addition on the active site histidine to the 3'-methylene.

PMN elastases: a comparison of the specificity of human isozymes and the enzyme from other species toward substrates and inhibitors.

The human elastases isolated from polymorphonuclear neutrophils (PMN) and purulent sputum displayed identical kinetic constants toward substrates and inhibitors. The elastases from the two sources yield identical N-terminal sequences and were recognized by antiserum prepared against human sputum elastase (HSE) isozyme-4 (I-4). The data support the proposal put forth by Twumasi and Liener (1977, J. Biol. Chem. 252, 1917-1926) that the human elastase from sputum is of PMN origin. PMN elastases from other species displayed kinetic constants toward both substrates and inhibitors significantly different from the human enzyme. Therefore, extrapolation of inhibitor profiles from these elastases to the human source should be avoided. Four groups of isozymes were resolved from HSE by FPLC. Only the most basic isozyme (I-4) was obtained as a single species. The isozymes displayed identical macroscopic kinetic constants toward several substrates and two classes of inhibitors. The similar partition ratios observed with a cephalosporin-derived inhibitor suggest that the microscopic rate constants are also identical. The data support the proposal suggested by Baugh and Travis (1976, Biochemistry 15, 836-841) that HLE isozymes differ only in carbohydrate content. Whatever the source of human PMN elastase heterogeneity, it does not result in heterogeneous catalytic properties. In addition, a new protein was identified in elastase preparations derived from human sputum. This protein displayed homology to serine proteases and properties suggesting that it is identical to azurocidin.

Inhibition of human leukocyte elastase. 2. Inhibition by substituted cephalosporin esters and amides.

A variety of 7 alpha-methoxycephalosporin ester and amide sulfones were prepared and tested to determine the structure-activity relations for inhibition of human leukocyte elastase (HLE), a serine protease which has been implicated in several degenerative lung and tissue diseases. The most potent IC50 values were obtained with neutral, lipophilic derivatives, with the esters being more active than the amides. However, the best time-dependent inhibition in this series was observed with the p- and m-carboxybenzyl esters 7b and 7c. These results are discussed in terms of the proposed mechanism of inhibition as well as a molecular modeling study using the recently solved X-ray crystal structure of HLE.

Inhibition of human leukocyte elastase. 3. Synthesis and activity of 3'-substituted cephalosporins.

Several 3'-substituted cephalosporin sulfones were synthesized from 3-(hydroxymethyl)cephalosporin, which was prepared by Ti(OiPr)4 hydrolysis of the corresponding acetate. A method was also developed to prepare a 3-vinylcephalosporin. Some of these compound were found to be potent time-dependent inhibitors of human leukocyte elastase (HLE). The HLE inhibitory activity was correlated with sigma 1 and it was concluded that the potency was determined by the electron-withdrawing ability as well as the size of the substituent. A mechanism for inhibition of HLE by cephalosporin sulfones is proposed.

Synthesis and structure-activity relationships of a novel class of 5-lipoxygenase inhibitors. 2-(Phenylmethyl)-4-hydroxy-3,5-dialkylbenzofurans: the development of L-656,224.

The synthesis of a series of 2-(phenylmethyl)-4-hydroxy-3,5-dialkylbenzofurans and their inhibitory effects against leukotriene biosynthesis and 5-lipoxygenase activity in vitro are described. Many compounds in this series were found to be potent inhibitors of LTB4 production by human polymorphonuclear leukocytes with IC50 values ranging from 7 to 100 nM. Structure-activity relationships of the series are presented. Within this series, 2-[(4'-methoxyphenyl)methyl]-4-hydroxy-3-methyl-5-propyl-7-chlorobenz ofuran (L-656,224) showed extremely potent activity, inhibiting leukotriene biosynthesis in intact human leukocytes (IC50 = 11 nM), as well as the 5-lipoxygenase reaction catalyzed by cell-free preparations from rat leukocytes (IC50 = 36 nM), human leukocytes (IC50 = 0.4 microM), and the purified enzyme from porcine leukocytes (IC50 = 0.4 microM). The compound also shows oral activity in a number of animal models in vivo.

Pharmacological profile of the substituted beta-lactam L-659,286: a member of a new class of human PMN elastase inhibitors.

Human polymorphonuclear leukocyte elastase (PMN elastase) is inhibited by L-659,286 (7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4- triaz-in-3-yl)thio]methyl]-5-thia-1-aza-6R-bicyclo[4.2.O]oct-2-ene -2- pyrrolidine carboxamide-5,-dioxide) with a Ki of 0.4 microM. This inhibition is time-dependent, rapid, and only slowly reversible, with a t1/2 of greater than 3 days at 25 degrees C. L-659,286 is also highly selective for PMN elastase, as it does not inhibit thrombin, trypsin, papain, plasmin, chymotrypsin, or cathepsin G. L-659,286 administered intratracheally inhibits lung damage caused by administration via the same route of human PMN elastase into hamsters. In marmosets, L-659,286 is cleared from blood very rapidly after an intravenous injection but is recovered in bronchoalveolar lavage fluid for several hours after intratracheal administration.

L-656,224 (7-chloro-2-[(4-methoxyphenyl)methyl]-3- methyl-5-propyl-4-benzofuranol): a novel, selective, orally active 5-lipoxygenase inhibitor.

L-656,224 (7-chloro-2-[(4-methoxyphenyl)methyl]-3-methyl-5-propyl-4-benzofuranol) was a potent inhibitor of leukotriene biosynthesis in intact rat and human leukocytes and CXBG mastocytoma cells (IC50 values, 18-240 nM) and of crude human leukocyte and highly purified porcine leukocyte 5-lipoxygenase (IC50 value, 4 X 10(-7) M). The selectivity of L-656,224 for 5-lipoxygenase was shown through the relative lack of activity of the compound on 12-lipoxygenase, 15-lipoxygenase, cyclooxygenase, catalase, and myeloperoxidase. The compound showed (i) oral activity against hyperalgesia induced in the rat paw by injection of yeast or platelet-activating factor, (ii) dyspnea in sensitized inbred rats induced by an aerosol of antigen, and (iii) bronchoconstriction induced by an aerosol of Ascaris in squirrel monkeys, suggesting a role for 5-lipoxygenase inhibitors in the treatment of asthma and peripheral pain.