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The structure−activity relationship at the C-terminal position of peptide-based inhibitors of the hepatitis C virus NS3 protease is presented. The observation that the N-terminal cleavage product (DDIVPC-OH) of a substrate derived from the NS5A/5B cleavage site was a competitive inhibitor of the NS3 protease was previously described. The chemically unstable cysteine residue found at the P1 position of these peptide-based inhibitors could be replaced with a norvaline residue, at the expense of a substantial drop in the enzymatic activity. The fact that an aminocyclopropane carboxylic acid (ACCA) residue at the P1 position of a tetrapeptide such as 1 led to a significant gain in the inhibitory enzymatic activity, as compared to the corresponding norvaline derivative 2, prompted a systematic study of substituent effects on the three-membered ring. We report herein that the incorporation of a vinyl group with the proper configuration onto this small cycle produced inhibitors of the protease with much improved in vitro potency. The vinyl-ACCA is the first reported carboxylic acid containing a P1 residue that produced NS3 protease inhibitors that are significantly more active than inhibitors containing a cysteine at the same position.

A comparative NMR conformational analysis of three distinct tetrapeptide inhibitors of the Hepatitis C NS3 protease that differ at the 4-aryloxy-substituted P2 proline position was undertaken. Specifically, transferred nuclear Overhauser effect experiments in combination with restrained systematic conformational searches were used to characterize the orientation of the P2 aryl substituents of these inhibitors when bound to the NS3 protease. Differences between free and bound conformations were also investigated. Analysis of the results allowed the design of a new P2 aromatic substituent, which significantly increased the potency of our inhibitors. The bound conformation of a specific competitive inhibitor having this novel P2 substituent is also described, along with a model of this inhibitor bound to the NS3 protease. This NS3 protease/inhibitor complex model also supports a hypothetical stabilization role for the P2 residue of the substrates and/or inhibitors and further elucidates the subtle details of the binding of the P2 residue of substrate-based inhibitors.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (Kd 1 μM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Starting from palinavir (1), our lead HIV protease inhibitor, we have discovered a new series of truncated analogues in which the P3-P2 quinaldic-valine portion of 1 was replaced by 2‘,6‘-dimethylphenoxyacetyl. With EC50's in the 1−2 nM range, some of these compounds are among the most potent inhibitors of HIV replication in vitro, reported to date. One of the most promising members in this series (compound 27, BILA 2185 BS) exhibited a favorable overall pharmacokinetic profile, with 61% apparent oral bioavailability in rat. X-ray crystal structures and molecular modeling were used to rationalize the high potency resulting from incorporation of this structurally simple, achiral ligand into the P3-P2 position of hydroxyethylamine-based HIV protease inhibitors.

p56^lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56^lck promotes phosphotyrosine-dependent protein−protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56^lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2−3 μM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced line-broadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and protease-bound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors.

A 3‐year experiment was conducted to monitor and subsequently predict field water contents using the water transport model. Saturated hydraulic conductivities and soil water characteristic curve results were measured and averaged for each soil layer. The Millington‐Quirk (MQ) method was used to calculate average model parameters. Direct use of the MQ‐derived conductivity and diffusivity functions did not give an exact fit to the observed field results. However, when reasonable adjustments were made to the K(θ) and D(θ) functions the prediction was improved considerably. The methodology for “averaging” and utilizing field data to derive model parameters, and subsequent adjustment or optimization of the model parameters all require more research if we hope to use the unsaturated flow equation for field water content prediction.

A simulation model describing water movement through field-cropped soils was developed. The soil water flow equation, which included a root extraction term, was approximated using a finite difference technique. Hydraulic conductivity (K) and diffusivity (D) functions were calculated by using the Millinton-Quirk technique. The chosen boundry conditions, rainfall, evapotranspiration, and a fluctuating water table approximated those existing in the field. Submodels to calculate interception, evaporation, and transpiration were included in the model. Model predictions were in good agreement with field data from a fairly uniform soil. On a soil containing discontinous clay varves the predictions were not satisfactory when the original calculated Kand Dfunctions were used. Predicted and measured water content profiles were in closer agreement when the Kand Dfunctions were modified within the limits of the measured moisture retention curve data.

The development of novel monobactam inhibitors of HCMV protease incorporating a carbon side chain at C-4 and a urea function at N-1 is described. Substitution with small groups at the C-3 position of the β-lactam ring gave an increase in enzymatic activity and in stability; however, a lack of selectivity against other serine proteases was noted. The use of both tri- and tetrasubstituted urea functionalities gave effective inhibitors of HCMV protease. Benzyl substitution of the urea moiety was beneficial, especially when strong electron-withdrawing groups where attached at the para position. Modest antiviral activity was found in a plaque reduction assay.

Results from a 2‐year study concerned with additions and losses of N from cropped fields in an intensively farmed sandy loam watershed in southern Ontario indicated that heavily fertilized hurley tobacco (Nicotiana tabacumL. [Kentucky 2110]) (220 kg N/ha per year) potato (Solanum tuberosumL. [Superior]) (165 kg N/ha per year) fields can potentially lose up to 52 and 92 kg N/ha per year, respectively, from the 75‐cm profile by leaching and denitrification. Mineral N production rates calculated from results obtained in the field over the growing season varied from 0.0 to 0.73 kg N/ha per year. The lower rate resulted from leaching losses. The monitored NO3‐N profile distributions under fertilized hurley tobacco and potato crops showed definite leaching patterns. Soil water samples taken periodically from the potato field at 90 and 150 cm showed NO3‐N moving through the lower profile in response to rainfall events. A plot treated with Cl showed no significant Cl leaching losses until early fall when rains moved about 45% of the added Cl below 75 cm.

Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide Ac-pYEEI binds to the SH2 domain of p56^lck (Lck) with an affinity of 0.1 μM. Starting from Ac-pYEEI, we have designed potent antagonists of the Lck SH2 domain which are reduced in peptidic character and in which the three carboxyl groups have been eliminated. The two C-terminal amino acids (EI) have been replaced by benzylamine derivatives and the pY + 1 glutamic acid has been substituted with leucine. The best C-terminal fragment identified, (S)-1-(4-isopropylphenyl)ethylamine, binds to the Lck SH2 domain better than the C-terminal dipeptide EI. Molecular modeling suggests that the substituents at the 4-position of the phenyl ring occupy the pY + 3 lipophilic pocket in the SH2 domain originally occupied by the isoleucine side chain. This new series of phosphotyrosine-containing dipeptides binds to the Lck SH2 domain with potencies comparable to that of tetrapeptide 1.

In field experiments conducted on a well-drained, sandy soil, 59 and 99 percent of the added chloride was lost by leaching from the 0–75-cm layer by the end of October in 1975 and 1976, respectively. Chloride losses were proportional to total precipitation. The average spring-to-fall rates of N losses for 1975 and 1976 were 0.65 and 1.45 kg N/ha/day, and were directly proportional to N fertilization rates of 120 and 255 kg/ha, respectively. NO3-N and Cl tended to show similar distribution patterns in the profile, and ratios of Cl to NO3-N indicated predominant leaching rather than denitrification losses. N and Cl losses appeared to be associated with irregular, diffuse bulges, rather than with distinct peaks of solute moving steadily downward.

Nitrate nitrogen and chloride concentrations were monitored in a 3‐year field study in order to examine factors affecting their variability. Fertilized and control plots, established each year, were sampled at least eight times throughout the year by removing about six‐replicate soil cores for individual analyses each sampling time. It was found that non‐uniform fertilization application accounted for 38 to 100% of the sampling variability immediately after fertilization. In fall and spring, differential rates of leaching of NO3‐N and Cl appeared to contribute significantly to variability increasing C.V.'s from about 40% to over 100%. Field and model results showed that differential leaching patterns could be attributed to two main factors: (i) variability of physical properties that determine water storage and transport, and (ii) point differences in redistribution of incoming rain (runoff) due to differences in surface microrelief.

Eleven agricultural watersheds in southern Ontario were sampled approximately 3,000 times during the years 1975–1977 for Total Kjeldahl N (TKN) and NO3‐N as part of the Canadian activities of the Pollution from Land Use Activities Reference Group (PLUARG). Only 12% of runoff samples had NO3‐N concentrations below 0.3 mg L−1, while 3% of the samples exceeded the 10‐mg L−1Ontario drinking water standard. Two‐year‐average total N stream loadings ranged from 3.5 to 29.2 kg N watershed‐ha−1. The NO3‐N loads averaged 75% of total N loads over all 11 watersheds. Both NO3‐N and total N unit‐area loads were significantly correlated with total N addition to the watershed, and with percent of watershed under cultivation, in row‐crops and corn (Zea maysL.), or tile‐drained. The TKN loads were positively correlated with watershed surface‐soil clay content and with extent of soils with high surface runoff potential. Remedial measures to reduce stream N loading should only be considered for watersheds where stream N loading much exceeds inputs of N from precipitation. In these watersheds, improved fertilizer N efficiency in corn production may provide the greatest potential for reduced stream N loading. Proper location of manure storage sites is most important to minimize N pollution from livestock activities. About 70% of both stream NO3‐N and total N loading occurred from January to April during the spring runoff season. Agronomic practices that reduce either postharvest teachable soil N or erosion during spring runoff merit future research.

Soil water retention curves were measured on cores taken from five sites at six depths in a Bainsville clay loam soil. There were differences in both the shape and magnitude of the average moisture characteristic curves from one location to another in the 225m2plot. Coefficients of variation ranged from 4.2 to 13 percent in the surface layers and from 2.4 to 6.5 percent in the deeper layers. There were no consistent trends in variability with respect to tensions from 0 to -500 cm H2O. Predicted and measured hydraulic conductivity functions were variable, often showing a 100-fold difference at a given water content. The measured Kfunctions were steeper than those predicted, and the match between them was considered reasonable only at the lower water contents.

The fusion-generating phage lambda plac Mu1 was used to produce fusions of lacZ to fhuA, the gene encoding the ferrichrome-iron receptor (FhuA protein) in the outer membrane of Escherichia coli K-12. Fusions to the fhuA gene in a delta (lac) strain were selected by their resistance to bacteriophage phi 80 vir. Ten independent (fhuA'-'lacZ) fusions were all Lac+ and were resistant to the lethal agents which require the FhuA protein as receptor, i.e., phi 80 vir, T5, T1, UC-1, and colicin M; none could utilize ferrichrome as the sole iron source. Specialized transducing phages were obtained by illegitimate excision from the chromosome of each of the fusion-bearing strains, and EcoRI fragments which encoded the fusions were subcloned into the high-copy plasmid pMLB524. Physical mapping of the fusion-containing plasmids confirmed the presence of three restriction sites which were also located on the chromosomal DNA of sequences near the fhuA gene. The direction of transcription of the fhuA gene was deduced from the direction of transcription of the (fhuA'-'lacZ) gene fusion. Identification of the chimeric proteins was made by both radiolabeling cells and immunoprecipitating the LacZ-containing proteins with antibody to beta-galactosidase and by preparing whole cell extracts from Lac+ cells containing the cloned gene fusions. Two sizes of (FhuA'-'LacZ) proteins were detected, 121 kDa and 124 kDa. The DNA sequences at the unique fusion joints were determined. The sequence information allowed us to identify three distinct fusion joints which were grouped as follows, type I fusions, 5'-ACT GCT CAG CCA A-3'; type IIa fusions, 5'-GCG GTT GAA CCG A-3'; and type IIb fusions: 5'-ACC GCT GCA CCT G-3'. To orient these fhuA fusion joints, the complete nucleotide sequence of the fhuA gene was determined from a 2,902-base-pair fragment of DNA. A single open reading frame was found which translated into a 747-amino acid polypeptide. The signal sequence of 33 amino acids was followed by a mature protein with a molecular weight of 78,992. Alignment of the amino acid sequence of the FhuA protein with the amino acid sequences presented for two other tonB-dependent receptor proteins in the outer membrane of E. coli showed an area of local homology at the amino terminus of all three proteins.